Compositions and methods of making expanded hematopoietic stem cells using derivatives of fluorene

ABSTRACT

This invention is directed to, inter alia, compounds, methods, systems, and compositions for the maintenance, enhancement, and expansion of hematopoietic stem cells derived from one or more sources of CD34+ cells. Sources of CD34+ cells include bone marrow, cord blood, mobilized peripheral blood, and non-mobilized peripheral blood. Also provided herein are compounds of Formula I 
     
       
         
         
             
             
         
       
     
     which are useful in maintaining, enhancing, and expanding of hematopoietic stem cells.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Patent ApplicationNo. 62/578,297, filed Oct. 27, 2017; and U.S. Provisional PatentApplication No. 62/583,328, filed Nov. 8, 2017, the entire contents ofeach are incorporated by reference herein in their entireties.

FIELD OF INVENTION

This invention is directed to, inter alia, methods and systems formaintaining and/or enhancing the expansion of hematopoietic stem cellsand/or progenitors in culture, media for culturing hematopoietic stemcells and progenitors, and therapeutic compounds and compositionscomprising the same for treatment of hematologic disorders.

BACKGROUND OF THE INVENTION

The maintenance of the hematopoietic system relies on primitivepluripotent hematopoietic stem cells (HSCs) that have the capacity toself-renew and repopulate all the blood cell lineages with relevantprogenitor cells. Due to their capacity for self-renewal and theirmultipotent, long term reconstituting potential, HSCs have long beenconsidered ideal for transplantation to reconstitute the hematopoieticsystem after treatment for various hematologic disorders or as a targetfor the delivery of therapeutic genes. Additionally, human HSCs havepotential applications for restoring the immune system in autoimmunediseases and in the induction of tolerance for allogenic solid organtransplantation.

The classical hematopoietic expansion cytokines thrombopoietin (TPO),stem cell factor (SCF), interleukin-3 (IL-3) and fms-related tyrosinekinase 3 ligand (FLT3L) are insufficient for the true maintenance andexpansion of HSCs. In these cultures, HSCs generally lose their potencywithin a week. Cord blood may be one of the best sources for HSCsavailable due to the relative potency of the cells and ease of access.Cord blood banks have extensive, preserved stocks of cells that can berapidly employed for therapeutic use. However, without extensiveexpansion of a single cord unit, each cord is unlikely to be used formore than one therapeutic dose or application.

Considering the therapeutic benefits that maintenance and expansion, orenhancement of HSCs and/or early hematopoietic progenitor cells wouldenable, it is critical that new, aggressive, efficient, yet safeprotocols and reagents be developed to meet this goal. The presentdisclosure addresses this need and provides related advantages as well.

Throughout this specification, various patents, patent applications andother types of publications (e.g., journal articles, electronic databaseentries, etc.) are referenced. The disclosure of all patents, patentapplications, and other publications cited herein are herebyincorporated by reference in their entirety for ail purposes.

SUMMARY

Provided herein, inter alia, are compounds, methods, and compositionsfor the rapid expansion, maintenance, and enhancement of hematopoieticstem cells and/or progenitors derived from one or more sources of CD34+cells.

Accordingly, in some aspects, provided herein are compounds of Formula I

wherein R¹, R², R³, R^(4a), R^(4b), A, m, and n are as defined below.

Additionally, in some aspects, provided herein are methods for expandinghematopoietic stem cells and/or progenitors in culture, the methodincluding contacting a source of CD34+ cells in culture with aneffective amount of a compound of Formula I, I-1, I-2, Ia, Ia′, Ia1,Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb,IIb1, IIc, IIa1, III, IIIa, IIIa′, or IIIa1 or a compound of Table 1,each of which are further described below. In some embodiments, themethod for expanding hematopoietic stem cells and progenitors in culturerestricts retinoic acid signaling. In some embodiments, retinoic acidsignaling is limited by using media that controls or reduces the amountof retinoic acid. In some embodiments, the media includes a retinoicacid receptor (RAR) inhibitor or modulator. In some embodiments, the RARinhibitor is ER50891.

In some aspects, source of CD34+ cells is bone marrow, cord blood,mobilized peripheral blood, or non-mobilized peripheral blood. In someaspects, the source of CD34+ cells is non-mobilized peripheral blood. Insome aspects, the source of CD34+ cells includes: (a) CD34+hematopoietic progenitors; (b) CD34+ early hematopoietic progenitorsand/or stem cells; (c) CD133+ early hematopoietic progenitors and/orstem cells; and/or (d) CD90+ early hematopoietic progenitors and/or stemcells.

In some aspects, the method stabilizes the hematopoietic stem cellphenotype. In some aspects, the hematopoietic stem cell phenotypeincludes: CD45+, CD34+, CD133+, CD90+, CD45RA−, CD38 low/−, and negativefor major hematopoietic lineage markers including CD2, CD3, CD4, CD5,CD8, CD14, CD16, CD19, CD20, CD56. In some aspects, CD133+ and/or CD90+positive cells are increased compared to cells in culture that are notcontacted with a compound of Formula I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa1, IIb, IIb1, IIc,IIc1, III, IIIa, IIIa′, or IIIa1 or a compound of Table 1. In someaspects, the cells exhibit at least about two times the number of CD133+and/or CD90+ positive cells compared to cells in culture that are notcontacted with a compound of Formula I or a subembodiment disclosedherein. In some aspects, CD90+ cells are increased compared to cells inculture that are not contacted with a compound of Formula I or asubembodiment disclosed herein. In some aspects, CD38 low/− cells areincreased compared to cells in culture that are not contacted with acompound of Formula I or a subembodiment disclosed herein. In someaspects, CD90+ and CD38 low/− cells are increased compared to cells inculture that are not contacted with a compound of Formula I or asubembodiment disclosed herein. In some aspects, the source of the CD34+cells is a human being.

In some aspects, provided herein are methods for producing a cellculture medium for culturing hematopoietic stem cells (HSC) and/orproginator cells. The method involves combining a base or a feed medium;and a compound of Formula I, I-1, I-2, Ia, Ia′, Ia1, Ia1′, Ia2, Ia2′,Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1, IIc, IIc1,III, IIIa, IIIa′, or IIIa1 or a compound of Table 1

In some aspects, provided herein are systems for maintaining and/orenhancing the expansion of hematopoietic stem cells in culture. Thissystem includes a source of CD34+ cells in culture (such as a CD34+cells from one or more of bone marrow, cord blood, mobilized peripheralblood, and non-mobilized peripheral blood) and any of the cell culturemedia compositions described herein.

In some aspects, provided herein are methods for treating an individualin need of hematopoietic reconstitution. The method involvesadministering to the individual a therapeutic agent containing any ofthe cultured HSCs derived according to the methods of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D illustrates the expansive effect measured for Compound 1.001(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.001. Thefold change is calculated as described in Example 33.

FIG. 2A-D illustrates the expansive effect measured for Compound 1.002(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.002. Thefold change is calculated as described in Example 33.

FIG. 3A-D illustrates the expansive effect measured for Compound 1.003(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.003. Thefold change is calculated as described in Example 33.

FIG. 4A-D illustrates the expansive effect measured for Compound 1.004(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.004. Thefold change is calculated as described in Example 33.

FIG. 5A-D illustrates the expansive effect measured for Compound 1.005(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.005. Thefold change is calculated as described in Example 33.

FIG. 6A-D illustrates the expansive effect measured for Compound 1.006(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.006. Thefold change is calculated as described in Example 33.

FIG. 7A-D illustrates the expansive effect measured for Compound 1.007(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.007. Thefold change is calculated as described in Example 33.

FIG. 8A-D illustrates the expansive effect measured for Compound 1.008(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.008. Thefold change is calculated as described in Example 33.

FIG. 9A-D illustrates the expansive effect measured for Compound 1.009(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.009. Thefold change is calculated as described in Example 33.

FIG. 10A-D illustrates the expansive effect measured for Compound 1.010(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.010. Thefold change is calculated as described in Example 33.

FIG. 11A-D illustrates the expansive effect measured for Compound 1.011(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.011. Thefold change is calculated as described in Example 33.

FIG. 12A-D illustrates the expansive effect measured for Compound 1.012(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.012. Thefold change is calculated as described in Example 33.

FIG. 13A-D illustrates the expansive effect measured for Compound 1.013(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.013. Thefold change is calculated as described in Example 33.

FIG. 14A-D illustrates the expansive effect measured for Compound 1.014(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.014. Thefold change is calculated as described in Example 33.

FIG. 15A-D illustrates the expansive effect measured for Compound 1.015(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.015. Thefold change is calculated as described in Example 33.

FIG. 16A-D illustrates the expansive effect measured for Compound 1.016(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.016. Thefold change is calculated as described in Example 33.

FIG. 17A-D illustrates the expansive effect measured for Compound 1.017(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.017. Thefold change is calculated as described in Example 33.

FIG. 18A-D illustrates the expansive effect measured for Compound 1.018(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.018. Thefold change is calculated as described in Example 33.

FIG. 19A-D illustrates the expansive effect measured for Compound 1.019(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.019. Thefold change is calculated as described in Example 33.

FIG. 20A-D illustrates the expansive effect measured for Compound 1.020(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.020. Thefold change is calculated as described in Example 33.

FIG. 21A-D illustrates the expansive effect measured for Compound 1.021(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.021. Thefold change is calculated as described in Example 33.

FIG. 22A-D illustrates the expansive effect measured for Compound 1.022(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.022. Thefold change is calculated as described in Example 33.

FIG. 23A-D illustrates the expansive effect measured for Compound 1.023(columns) and controls: basic conditions (thin dashed lines) and +SFconditions (thick dashed lines). The data is reported as the fold changefrom days 2 to 7 for all live cells (A), CD34+ cells (B), CD34+/CD133+cells (C), and CD34+/CD133+/CD90+ cells (D). Each column reports thefold change in cells at the noted concentration of Compound 1.023. Thefold change is calculated as described in Example 33.

FIG. 24A-E report flow cytometric cell counts in cord blood samplescultured in “Base conditions” (white column, on left); “+SF Conditions”(diagonally hashed column, second from the left); “+1.008 conditions”(black column, second from the right); “+1.008/+ER conditions”(horizontally striped column, on the right). FIG. 24A reports the totalnumber of live cells in culture, and FIGS. 24B, 24C, 24D, and 24E showthat +1.008 and +1.008/+ER conditions increase the total number of CD34+cells (24B), CD34+/CD133+ cells (24C), CD34+/CD133+/CD90+(24D), andCD34+/CD133+/CD90+/CD38′ cells (24E).

FIG. 25A-E report the fold change in cell counts from day 2 to theindicated day based on the cord blood data reported in FIG. 24. “Baseconditions” (white column, on left); “+SF Conditions” (diagonally hashedcolumn, second from the left); “+1.008 conditions” black column, secondfrom the right); “+1.008/+ER conditions” (horizontally striped column,on the right) FIG. 25A reports the fold change of live cells in culture,and FIGS. 25B, 25C, 25D, and 25E show the fold change in the totalnumber of CD34+ cells (25B), CD34+/CD133+ cells (25C),CD34+/CD133+/CD90+(25D), and CD34+/CD133+/CD90+/CD38^(low/−) cells(25E).

FIG. 26A-D illustrates the expansive effect measured for Compound 1.005and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.005. The fold change is calculated as described in Example35.

FIG. 27A-D illustrates the expansive effect measured for Compound 1.006and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.006. The fold change is calculated as described in Example35.

FIG. 28A-D illustrates the expansive effect measured for Compound 1.007and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.007. The fold change is calculated as described in Example35.

FIG. 29A-D illustrates the expansive effect measured for Compound 1.008and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.008. The fold change is calculated as described in Example35.

FIG. 30A-D illustrates the expansive effect measured for Compound 1.009and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.009. The fold change is calculated as described in Example35.

FIG. 31A-D illustrates the expansive effect measured for Compound 1.010and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.010. The fold change is calculated as described in Example35.

FIG. 32A-D illustrates the expansive effect measured for Compound 1.013and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.013. The fold change is calculated as described in Example35.

FIG. 33A-D illustrates the expansive effect measured for Compound 1.014and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.014. The fold change is calculated as described in Example35.

FIG. 34A-D illustrates the expansive effect measured for Compound 1.015and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.015. The fold change is calculated as described in Example35.

FIG. 35A-D illustrates the expansive effect measured for Compound 1.021and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.021. The fold change is calculated as described in Example35.

FIG. 36A-D illustrates the expansive effect measured for Compound 1.022and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.022. The fold change is calculated as described in Example35.

FIG. 37A-D illustrates the expansive effect measured for Compound 1.023and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.023. The fold change is calculated as described in Example35.

FIG. 38A-D illustrates the expansive effect measured for Compound 1.024and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.024. The fold change is calculated as described in Example35.

FIG. 39A-D illustrates the expansive effect measured for Compound 1.025and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.025. The fold change is calculated as described in Example35.

FIG. 40A-D illustrates the expansive effect measured for Compound 1.026and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.026. The fold change is calculated as described in Example35.

FIG. 41A-D illustrates the expansive effect measured for Compound 1.027and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.027. The fold change is calculated as described in Example35.

FIG. 42A-D illustrates the expansive effect measured for Compound 1.028and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.028. The fold change is calculated as described in Example35.

FIG. 43A-D illustrates the expansive effect measured for Compound 1.029and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.029. The fold change is calculated as described in Example35.

FIG. 44A-D illustrates the expansive effect measured for Compound 1.030and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.030. The fold change is calculated as described in Example35.

FIG. 45A-D illustrates the expansive effect measured for Compound 1.031and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.031. The fold change is calculated as described in Example35.

FIG. 46A-D illustrates the expansive effect measured for Compound 1.032and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.032. The fold change is calculated as described in Example35.

FIG. 47A-D illustrates the expansive effect measured for Compound 1.033and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.033. The fold change is calculated as described in Example35.

FIG. 48A-D illustrates the expansive effect measured for Compound 1.034and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.034. The fold change is calculated as described in Example35.

FIG. 49A-D illustrates the expansive effect measured for Compound 1.035and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.035. The fold change is calculated as described in Example35.

FIG. 50A-D illustrates the expansive effect measured for Compound 1.036and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.036. The fold change is calculated as described in Example35.

FIG. 51A-D illustrates the expansive effect measured for Compound 1.037and “cytokines only” control (dashed lines). The data is reported as thefold change from days 1 to 7 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D). Each datapoint reports the fold change in cells at the noted concentration ofCompound 1.037. The fold change is calculated as described in Example35.

FIG. 52A-F illustrates the expansive effect measured for Compound 1.010(black bars) and “cytokines only” control (white bars) after 7, 10, 14,and 21 days in culture using hematopoietic stem cells derived from cordblood. The data is reported as the fold change from day 1 to theindicated number of days for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), CD34+/CD133+/CD90+ cells (D),CD34+/CD13+/CD90+/CD38^(low/−) cells (E), and CD34+/CD13+/CD90+/CD45RA−cells (F).

FIG. 53A-F illustrates the expansive effect measured for Compound 1.010(black bars) and “cytokines only” control (white bars) after 7, 10, 14,and 21 days in culture using hematopoietic stem cells derived frommobilized peripheral blood. The data is reported as the fold change fromday 1 to the indicated number of days for all live cells (A), CD34+cells (B), CD34+/CD133+ cells (C), CD34+/CD133+/CD90+ cells (D),CD34+/CD13+/CD90+/CD38^(low/−) cells (E), and CD34+/CD13+/CD90+/CD45RA−cells (F).

FIG. 54A-F illustrates the expansive effect measured for Compound 1.010(black bars) and “cytokines only” control (white bars) after 7, 10, 14,and 21 days in culture using hematopoietic stem cells derived fromnon-mobilized peripheral blood. The data is reported as the fold changefrom day 1 to the indicated number of days for all live cells (A), CD34+cells (B), CD34+/CD133+ cells (C), CD34+/CD133+/CD90+ cells (D),CD34+/CD13+/CD90+/CD38^(low/−) cells (E), and CD34+/CD13+/CD90+/CD45RA−cells (F).

FIG. 55A-D illustrates the expansive effect measured for Compound 1.010(black bars) and “cytokines only” control (white bars) after 9 days inculture at atmospheric oxygen. The data is reported as the fold changefrom day 1 to day 9 for all live cells (A), CD34+ cells (B),CD34+/CD133+ cells (C), and CD34+/CD133+/CD90+ cells (D).

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein provides, inter alia, compounds,compositions, and methods of using the same for the maintenance,enhancement, and expansion of hematopoietic stem cells (HSCs). Themethods and compositions for the maintenance, enhancement, and expansionof hematopoietic stem cells (HSCs) can be derived from one or moresources of CD34+ cells (such as, non-mobilized peripheral blood).Sources of CD34+ cells can include peripheral blood, cord blood, andbone marrow. Peripheral blood is known to reliably carry a small numberof CD34+ hematopoietic progenitors and an even smaller number of CD34+and CD133+ early hematopoietic progenitors and stem cells. Being thesource with the least potent, least enriched, most dilute andimpractically small numbers of apparent stem cells by nature, stem cellscientists have generally concluded that this source is unlikely to betherapeutically relevant compared to other potential sources of HSCs,such as bone marrow cells, mobilized peripheral blood, cord blood, andeven embryonic or induced pluripotent stem cell (also known asiPS)-sourced CD34+ cells. Despite failed efforts to expand blood stemcells using more potent sources of cells, such as bone marrow and cordblood, there is some evidence that mitogenic, survival promoting, andquiescence inducing factors can impact the phenotype of these cells inpositive ways and even help maintain them for some time in vitro.

The inventors of the present invention have observed that multipotentblood stem cells and progenitors can be successfully maintained,expanded, and enhanced by culturing these cells in a medium containing aCompound of Formula I, I-1, I-2, Ia, Ia′, Ia1, Ia1′, Ia2, Ia2′, Ib, Ib1,Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1, IIc, IIa, III, IIIa,IIIa′, or IIIa1, or a compound of Table 1, each of which are furtherdescribed below. In particular, the methods and compositions of thepresent invention are not only able to successfully derive HSCs fromconventional sources, such as bone marrow, cord blood, and mobilizedperipheral blood, but also from non-conventional sources such asnon-mobilized peripheral blood. As such, the methods and compositionsdescribed herein provide for the generation of a therapeuticallyrelevant stem cell transplant product derived from an easy to access andpermanently available tissue source, without the need to expose thedonor to significant risk or pain and which is more readily availablethan cord blood.

I. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,cell biology, biochemistry, nucleic acid chemistry, and immunology,which are well known to those skilled in the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, fourth edition (Sambrook et al., 2012) and MolecularCloning: A Laboratory Manual, third edition (Sambrook and Russel, 2001),(jointly referred to herein as “Sambrook”); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987, includingsupplements through 2014); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Antibodies: A Laboratory Manual, Second edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(Greenfield, ed., 2014), Beaucage et al. eds., Current Protocols inNucleic Acid Chemistry, John Wiley & Sons, Inc., New York, 2000,(including supplements through 2014), Gene Transfer and Expression inMammalian Cells (Makrides, ed., Elsevier Sciences B.V., Amsterdam,2003), and Current Protocols in Immunology (Horgan K and S. Shaw (1994)(including supplements through 2014).

II. Definitions

Hematopoietic cells encompass not only HSCs, but also erythrocytes,neutrophils, monocytes, platelets, megakaryocytes, mast cells,eosinophils and basophils, B and T lymphocytes and NK cells as well asthe respective lineage progenitor cells.

As used herein, “maintaining the expansion” of HSCs refers to theculturing of these cells such that they continue to divide rather thanadopting a quiescent state and/or losing their multipotentcharacteristics. Multipotency of cells can be assessed using methodsknown in the art using known multipotency markers. Exemplarymultipotency markers include CD133+, CD90+, CD38 low/−, CD45RAnegativity but overall CD45 positivity, and CD34. In some examples, CD34low/− cells may be hematopoietic stem cells. In examples, where CD34low/− cells are hematopoietic stem cells, these cells express CD133.

As used herein the term “cytokine” refers to any one of the numerousfactors that exert a variety of effects on cells, for example, inducinggrowth or proliferation. The cytokines may be human in origin, or may bederived from other species when active on the cells of interest.Included within the scope of the definition are molecules having similarbiological activity to wild type or purified cytokines, for exampleproduced by recombinant means; and molecules which bind to a cytokinefactor receptor and which elicit a similar cellular response as thenative cytokine factor.

The term “culturing” refers to the propagation of cells on or in media(such as any of the media disclosed herein) of various kinds.

As used herein, the term “mobilized blood” refers to cells which havebeen exposed to an agent that promotes movement of the cells from thebone marrow into the peripheral blood and/or other reservoirs of thebody (e.g., synovial fluid) or tissue.

As used herein, the phrase “non-mobilized peripheral blood” refers to ablood sample obtained from an individual who has not been exposed to anagent that promotes movement of the cells from the bone marrow into theperipheral blood and/or other reservoirs of the body. In some cases,“non-mobilized peripheral blood” refers to the blood from an individualwho has not been exposed to an agent that promotes movement of the cellsfrom the bone marrow into the peripheral blood and/or other reservoirsof the body for at least 1, 3, 5, 7, or 10 or more days. In some cases,“non-mobilized peripheral blood” refers to the blood of individuals whohave not been exposed to an agent that promotes movement of the cellsfrom the bone marrow into the peripheral blood and/or other reservoirsof the body for at least 5, 7, 10, 14, 21 or more days. In some cases,“non-mobilized peripheral blood” refers to the blood of individuals whohave not been exposed to an agent that promotes movement of the cellsfrom the bone marrow into the peripheral blood and/or other reservoirsof the body for at least 14, 21, 28, 35, 42, 49 or more days.

“Tetraspanins,” (also called “tetraspans” or “the transmembrane 4superfamily” (TM4SF)) as used herein, refer to a family of membraneproteins found in all multicellular eukaryotes that have fourtransmembrane domains, intracellular N- and C-termini and twoextracellular domains: one called the small extracellular domain or loop(SED/SEL or EC1) and the other, longer (typically 100 amino acidresidue), domain called the large extracellular domain/loop (LED/LEL orEC2). There are 34 tetraspanins in mammals, 33 of which have also beenidentified in humans. Tetraspanins display numerous properties thatindicate their physiological importance in cell adhesion, motility,activation and proliferation, as well as their contribution topathological conditions such as metastasis or viral infection.

An “individual” can be a vertebrate, a mammal, or a human. Mammalsinclude, but are not limited to, farm animals, sport animals, pets,primates, mice and rats. In one aspect, an individual is a human.

“Treatment,” “treat,” or “treating,” as used herein covers any treatmentof a disease or condition of a mammal, for example, a human, andincludes, without limitation: (a) preventing the disease or conditionfrom occurring in a subject which may be predisposed to the disease orcondition but has not yet been diagnosed as having it; (b) inhibitingthe disease or condition, i.e., arresting its development; (c) relievingand or ameliorating the disease or condition, i.e., causing regressionof the disease or condition; or (d) curing the disease or condition,i.e., stopping its development or progression. The population ofindividuals treated by the methods of the invention includes individualssuffering from the undesirable condition or disease, as well asindividuals at risk for development of the condition or disease.

“Alkyl” refers to a straight or branched, saturated, aliphatic radicalhaving the number of carbon atoms indicated. Alkyl can include anynumber of carbons, such as C₁₋₂, C₁₋₃, C₁₋₄, C₁₋₅, C₁₋₆, C₁₋₇, C₁₋₈,C₁₋₉, C₁₋₁₀, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₃₋₄, C₃₋₅, C₃₋₆, C₄₋₅, C₄₋₆ andC₅₋₆. For example, C₁₋₆ alkyl includes, but is not limited to, methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, hexyl, etc. Alkyl groups can be substituted orunsubstituted.

“Alkylene” refers to a straight or branched, saturated, aliphaticradical having the number of carbon atoms indicated, and linking atleast two other groups, i.e., a divalent hydrocarbon radical. The twomoieties linked to the alkylene can be linked to the same atom ordifferent atoms of the alkylene group. For instance, a straight chainalkylene can be the bivalent radical of —(CH₂)_(n)—, where n is 1, 2, 3,4, 5 or 6. Representative alkylene groups include, but are not limitedto, methylene, ethylene, propylene, isopropylene, butylene, isobutylene,sec-butylene, pentylene and hexylene. Alkylene groups can be substitutedor unsubstituted.

“Alkenyl” refers to a straight chain or branched hydrocarbon having atleast 2 carbon atoms and at least one double bond. Alkenyl can includeany number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇, C₂₋₈,C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, and C₆.Alkenyl groups can have any suitable number of double bonds, including,but not limited to, 1, 2, 3, 4, 5 or more. Examples of alkenyl groupsinclude, but are not limited to, vinyl (ethenyl), propenyl, isopropenyl,1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl,isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl,2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can be substitutedor unsubstituted.

“Alkynyl” refers to either a straight chain or branched hydrocarbonhaving at least 2 carbon atoms and at least one triple bond. Alkynyl caninclude any number of carbons, such as C₂, C₂₋₃, C₂₋₄, C₂₋₅, C₂₋₆, C₂₋₇,C₂₋₈, C₂₋₉, C₂₋₁₀, C₃, C₃₋₄, C₃₋₅, C₃₋₆, C₄, C₄₋₅, C₄₋₆, C₅, C₅₋₆, andC₆. Examples of alkynyl groups include, but are not limited to,acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl,butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl,1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl,1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl.Alkynyl groups can be substituted or unsubstituted.

“Halogen” or “halo” refers to fluorine, chlorine, bromine and iodine.

“Haloalkyl” refers to alkyl, as defined above, where some or all of thehydrogen atoms are replaced with halogen atoms. As for alkyl group,haloalkyl groups can have any suitable number of carbon atoms, such asC₁₋₆. For example, haloalkyl includes trifluoromethyl, fluoromethyl,etc. In some instances, the term “perfluoro” can be used to define acompound or radical where all the hydrogens are replaced with fluorine.For example, perfluoromethyl refers to 1,1,1-trifluoromethyl.

“Alkoxy” refers to an alkyl group having an oxygen atom that connectsthe alkyl group to the point of attachment: alkyl-O—. As for alkylgroup, alkoxy groups can have any suitable number of carbon atoms, suchas C₁₋₆. Alkoxy groups include, for example, methoxy, ethoxy, propoxy,iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,pentoxy, hexoxy, etc. The alkoxy groups can be further substituted witha variety of substituents. Alkoxy groups can be substituted orunsubstituted.

“Oxo” refers to an oxygen atom that is linked to the remainder of acompound with a double bonded (e.g.

wherein the “wavy line” (

) denotes the point of attachment to the remainder of the molecule).

“Oxime” refers to an nitrogen atom that is linked to the remainder of acompound with a double bonded and includes a further covalent bond to ahydroxyl moiety (e.g.

wherein the “wavy line” (

) denotes the point of attachment to the remainder of the molecule).

“Hydroxyalkyl” refers to an alkyl group, as defined above, where atleast one of the hydrogen atoms is replaced with a hydroxy group. As forthe alkyl group, hydroxyalkyl groups can have any suitable number ofcarbon atoms, such as C₁₋₆. Exemplary hydroxyalkyl groups include, butare not limited to, hydroxy-methyl, hydroxyethyl (where the hydroxy isin the 1- or 2-position), hydroxypropyl (where the hydroxy is in the 1-,2- or 3-position), hydroxybutyl (where the hydroxy is in the 1-, 2-, 3-or 4-position), hydroxypentyl (where the hydroxy is in the 1-, 2-, 3-,4- or 5-position), hydroxyhexyl (where the hydroxy is in the 1-, 2-, 3-,4-, 5- or 6-position), 1,2-dihydroxyethyl, and the like.

“Heteroaryl” refers to a monocyclic ring assembly containing 5 to 6 ringatoms, where from 1 to 3 of the ring atoms are a heteroatom such as N, Oor S. Additional heteroatoms can also be useful, including, but notlimited to, B, Al, Si and P. The heteroatoms can also be oxidized, suchas, but not limited to, —S(O)— and —S(O)₂—. The heteroaryl group caninclude groups such as pyrrole, pyridine, imidazole, pyrazole, triazole,tetrazole, pyrazine, pyrimidine, pyridazine, triazine (1,2,3-, 1,2,4-and 1,3,5-isomers), thiophene, furan, thiazole, isothiazole, oxazole,and isoxazole. Heteroaryl groups can be substituted or unsubstituted.

“Heterocycloalkyl” refers to a saturated ring system having from 3 to 6ring members and from 1 to 3 heteroatoms of N, O and S. Additionalheteroatoms can also be useful, including, but not limited to, B, Al, Siand P. The heteroatoms can also be oxidized, such as, but not limitedto, —S(O)— and —S(O)₂—. Any suitable number of heteroatoms can beincluded in the heterocycloalkyl groups, such as 1, 2, 3, or 1 to 2, 1to 3, 2 to 3. The heterocycloalkyl group can include groups such asaziridine, azetidine, pyrrolidine, piperidine, azepane, azocane,quinuclidine, pyrazolidine, imidazolidine, piperazine (1,2-, 1,3- and1,4-isomers), oxirane, oxetane, tetrahydrofuran, oxane(tetrahydropyran), oxepane, thiirane, thietane, thiolane(tetrahydrothiophene), thiane (tetrahydrothiopyran), oxazolidine,isoxazolidine, thiazolidine, isothiazolidine, dioxolane, dithiolane,morpholine, thiomorpholine, dioxane, or dithiane. Heterocycloalkylgroups can be unsubstituted or substituted. For example,heterocycloalkyl groups can be substituted with C₁₋₆ alkyl or oxo (═O),among many others.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, unrecited elements or methodsteps. By contrast, the transitional phrase “consisting of” excludes anyelement, step, or ingredient not specified in the claim. Thetransitional phrase “consisting essentially of” limits the scope of aclaim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains.

As used herein, the singular terms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomer,geometric isomers, regioisomers and individual isomers (e.g., separateenantiomers) are all intended to be encompassed within the scope of thepresent invention. In some embodiments, the compounds of the presentinvention are a particular enantiomer or diastereomer substantially freeof other forms.

The term “substantially free” refers to an amount of 10% or less ofanother form, preferably 8%, 5%, 4%, 3%, 2%, 1%, 0.5%, or less ofanother form. In some embodiments, the isomer is a stereoisomer.

III. Compositions of the Invention

Provided herein are cell cultures of expanded hematopoietic stem cells(HSC), cell culture media for maintaining and/or enhancing the expansionof hematopoietic stem cells in culture, and populations of cellscontaining HSCs. Such populations of cells containing HSCs can be madefrom the methodology described herein. Hematopoietic stem cell caninclude mammalian and avian hematopoietic stem cells. A population ofhematopoietic cells can have the potential for in vivo therapeuticapplication. The medium includes a base medium or a feed medium as wellas a compound of Formula I. Any suitable base or feed medium forculturing mammalian cells can be used in the context of the presentinvention and can include, without limitation, such commerciallyavailable media as DMEM medium, IMDM medium, StemSpan Serum-FreeExpansion Medium (SFEM), 199/109 medium, Ham's F10/F12 medium, McCoy's5A medium, Alpha MEM medium (without and with phenol red), and RPMI 1640medium. In some embodiments, the base or feed medium is Alpha MEM medium(without phenol red).

In some embodiments, the methods, media, systems, and kits providedherein do not include a tetraspanin. In some embodiments, the methods,media, systems, and kits provided herein also include a retinoic acidreceptor (RAR) inhibitor or modulator. In some embodiments, the RARinhibitor is ER50891.

Populations of cells containing HSCs provided herein confer the same orsimilar advantages of stem cells found in cord blood. A person of skillin the art would readily recognize the characteristics of stem cellsfrom cord blood and the advantageous properties therein. In someembodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% of the populations of cells containing HSCs provided herein areexpanded HSC cells. In some embodiments, the expanded HSC cells in thepopulations of cells have retained their stem cell phenotype for anextended period of time. For example, in some embodiments, populationsof cells containing HSCs include expanded HSC cells with cell surfacephenotypes that include CD45+, CD34+, CD133+, CD90+, CD45RA−, and/orCD38 low/− and have been cultured in vitro for at least 3, 7, 10, 13,14, 20, 25, 30, 40, or 50 or more days. In some embodiments, populationsof cells containing HSCs include expanded HSC cells with cell surfacephenotypes that includes CD133+ and/or CD90+ and have been cultured invitro for at least 3, 7, 10, 13, 19, 21 or more days.

A. Compounds of Formula

In one aspect, provided herein are compounds of Formula I

or a pharmaceutically acceptable salt, hydrate, or solvate thereof;wherein

-   -   A is a fused cyclic moiety selected from the group consisting of        a phenyl, C₃₋₆ cycloalkyl, heterocycloalkyl, and heteroaryl, or        is absent;        -   wherein each heterocycloalkyl comprises from 3 to 6 ring            members having 1 to 3 nitrogen atom ring members, and        -   each heteroaryl comprises 5 to 6 ring members having 1 to 3            nitrogen atom ring members;    -   R¹ is selected from the group consisting of —C(O)—NR^(b)—R^(1a),        —NR^(b)—C(O)—R^(1a), —NR^(b)—C(O)—R^(b), —NR^(b)—X¹—C(O)—R^(1a),        —C(O)—X¹—NR^(b)—R^(1a), —X¹—C(O)—NR^(b)—R^(1a),        —X¹—NR^(b)—C(O)—R^(1a), —NR^(b)—C(O)—X¹—C(O)—R^(1b),        —C(O)—NR^(b)—X¹—C(O)—R^(1b), —NR^(b)—C(O)—O—R^(1a),        —O—C(O)—NR^(b)—R^(1a), —X¹—NR^(b)—C(O)—O—R^(1a),        —X¹—O—C(O)—NR^(b)—R^(1a), —NR^(b)—R^(1a), —C(O)—R^(1a),        —O—C(O)—R^(1a), halo, and —NO₂;    -   R^(1a) is selected from the group consisting of H, C₁₋₁₀ alkyl;        C₁₋₁₀ haloalkyl;    -   R^(1b) is selected from the group consisting of —OR^(a),        —NR^(a)R^(b), heterocycloalkyl, and phenyl        -   wherein each heterocycloalkyl comprises from 5 to 6 ring            members having 1 to 3 heteroatom ring members selected from            the group consisting of nitrogen, oxygen, and sulfur, and        -   each heterocycloalkyl and phenyl is unsubstituted or            substituted with one to four C₁₋₄ alkyl, —OH, and halo;    -   each R² is independently selected from the group consisting of        halogen, —CN, —C₁₋₈ alkyl, —C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₁₋₈        haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈ alkoxy, —C(O)—R^(2a),        —NR^(b)—C(O)—R^(2a), —SR^(a), —X¹—SR^(a), —OR^(a), —X¹—OR^(a),        —NR^(a)R^(b), —X¹—NR^(a)R^(b), —S(O)₂R^(a), —S(O)₂NR^(a)R^(b),        —X¹—S(O)₂R^(a), —X¹—S(O)₂NR^(a)R^(b), and —O—C(O)—R    -   each R³ is independently selected from the group consisting of        halogen, —CN, —C₁₋₈ alkyl, —C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₁₋₈        haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈ alkoxy, —C(O)—R^(3a), —SR^(a),        —X¹—SR^(a), —OR^(a), —X¹—OR^(a), —NR^(a)R^(b), —X¹—NR^(a)R^(b),        —S(O)₂R^(a), —S(O)₂NR^(a)R^(b), —X¹—S(O)₂R^(a), and        —X¹—S(O)₂NR^(a)R^(b);    -   each R^(2a) and R^(3a) is independently selected from the group        consisting of H, C₁₋₁₀ alkyl, C₁₋₁₀ haloalkyl, —OR^(a),        —X¹—OR^(a), —NR^(a)R^(b), and —X¹—NR^(a)R^(b);    -   R^(4a) is selected from the group consisting of —OR^(a),        —NR^(a)R^(b), —O—C(O)—R^(a), and cyano;    -   R^(4b) is H; or R^(4a) and R^(4b) are combined to form an oxo or        an oxime moiety;    -   each R^(a) and R^(b) is independently selected from the group        consisting of H and C₁₋₄ alkyl;    -   each X¹ is C₁₋₄ alkylene;    -   the subscript n is an integer from 0 to 3; and    -   the subscript m is an integer from 0 to 2.

In some embodiments, the compound of Formula I is not2-fluoro-9H-fluoren-9-one, 2-amino-9H-fluoren-9-one,2-nitro-9H-fluoren-9-one, N-(9-oxo-9H-fluoren-2-yl)acetamide.

In some aspects, compounds of Formula I can inhibit or alter theactivity of PTEN, thereby providing improved conditions for expandingand maintaining hematopoietic stem cells in culture.

PTEN is known as a tumor suppressor that is mutated in a high frequencyof cancers. This protein negatively regulates intracellular levels ofphosphatidylinositol-3,4,5-trisphosphate (PIP₃) and functions as a tumorsuppressor by negatively regulating Akt/PKB signaling pathway. Aninhibitor of PTEN is a compound that decreases, blocks, prevents, orotherwise reduces the natural activity of PTEN.

In some embodiments, the compound of Formula I has the structure ofFormula I-1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein

-   -   R^(4a) is selected from the group consisting of —OR^(a), and        —NR^(a)R^(b);    -   R^(4b) is H.

In some embodiments, the compound of Formula I has the structure ofFormula I-2

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein

-   -   R^(4a) is selected from the group consisting of —OR^(a), and        —NR^(a)R^(b);    -   R^(4b) is H.

In some embodiments, A in Formula I, I-1, and 1-2 is

-   -   a fused cyclic moiety selected from the group consisting of a        C₃₋₆ cycloalkyl, heterocycloalkyl, and phenyl,    -   wherein each heterocycloalkyl comprises from 3 to 6 ring members        having 1 to 3 nitrogen atom ring members.

In some embodiments, A in Formula I, 1-2, and I-2 is

-   -   a fused cyclic moiety selected from the group consisting of a        C₃₋₆ cycloalkyl and phenyl.

In some embodiments, A in Formula I, 1-2, and I-2 is

-   -   a fused c C₃₋₆ cycloalkyl.

In some embodiments, R^(4a) in Formula I is —OR^(a); R^(4b) is H; orR^(4a) and R^(4b) are combined to form an oxo moiety.

In some embodiments, R^(4a) in Formula I is —OR^(a); R^(4b) is H.

In some embodiments, R^(4a) in Formula I is —NR^(a)R^(b); R^(4b) is H.

In some embodiments, the compound of Formula I has the structure ofFormula Ia

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula Ia has the structure ofFormula Ia′

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula Ia has the structure ofFormula Ia1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

In some embodiments, the compound of Formula Ia1 has the structure ofFormula Ia1′

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compounds of Formula Ia has the structure ofFormula Ia2.

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

In some embodiments, the compounds of Formula Ia2 has the structure ofFormula Ia2′.

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein

In some embodiments, the compound of Formula I has the structure ofFormula Ib

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compounds of Formula Ib has the structure ofFormula Ib1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

In some embodiments, the compounds of Formula Ib has the structure ofFormula Ib2.

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

In some embodiments, the compound of Formula I has the structure ofFormula Ic

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compounds of Formula Ic has the structure ofFormula Ic1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

In some embodiments, the compounds of Formula Ic has the structure ofFormula Ic2.

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

In some embodiments, the compound of Formula I has the structure ofFormula II

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula II has the structure ofFormula IIa

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula IIa has the structure ofFormula IIa′

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula IIa has the structure ofFormula IIa1

In some embodiments, the compound of Formula II has the structure ofFormula IIb

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula IIb has the structure ofFormula IIb1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula II has the structure ofFormula IIc

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula IIc has the structure ofFormula IIc1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula I has the structure ofFormula II

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula II has the structure ofFormula IIa

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula IIa has the structure ofFormula IIa′

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula IIa has the structure ofFormula IIa1

In some embodiments, the compound of Formula I has the structure ofFormula III

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula III has the structure ofFormula IIIa

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula IIIa has the structure ofFormula IIIa′

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula IIIa has the structure ofFormula IIIa1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, R¹ in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is selected from the groupconsisting of —C(O)—NR^(b)—R^(1a), —NR^(b)—C(O)—R^(1a),—NR^(b)—X¹—C(O)—R^(1a), —C(O)—X¹—NR^(b)—R^(1a), —X¹—C(O)—NR^(b)—R^(1a),—X¹—NR^(b)—C(O)—R^(1a), —NR^(b)—C(O)—X¹—C(O)—R^(1b),—C(O)—NR^(b)—X¹—C(O)—R^(1b), —NR^(b)—C(O)—O—R^(1a),—O—C(O)—NR^(b)—R^(1a), —NR^(b)—R^(1a), and —C(O)—R^(1a).

In some embodiments, R¹ in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is selected from the groupconsisting of —C(O)—NH—R^(1a), —NH—C(O)—R^(1a), —NH—C(O)—O—R^(1a),—O—C(O)—NH—R^(1a), —NH—R^(a), and —C(O)—R^(1a).

In some embodiments, R¹ in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is selected from the groupconsisting of —NH—C(O)—R^(a), —NH—C(O)—O—R^(a), and —NR^(b)—R^(1a).

In some embodiments, R¹ in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is selected from the groupconsisting of —NH—C(O)—R^(1a), and —NH—C(O)—O—R^(1a).

In some embodiments, R¹ in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, III, III, IIIa, IIIa′, or IIIa1 is —NH—C(O)—R^(1a).

In some embodiments, R¹ in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc, III, IIIa, IIIa′, or IIIa1 is halo.

In some embodiments, R¹ in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is fluorine.

In some embodiments, each R² in Formulas I, I-1, I-2, Ia, Ia′, Ia1,Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb,IIb1, IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is independently selectedfrom the group consisting of halogen, —C₁₋₈ alkyl, C₁₋₈ haloalkyl, —C₁₋₈alkoxy, —X¹—C₁₋₈ alkoxy, —C(O)—R^(2a), —NR^(b)—C(O)—R^(2a), —SR^(a),—X¹—SR^(a), —OR^(a), —X¹—OR^(a), —NR^(a)R^(b), and —X¹—NR^(a)R^(b).

In some embodiments, each R² in Formulas I, I-1, I-2, Ia, Ia′, Ia1,Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb,IIb1, IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is independently selectedfrom the group consisting of halogen, —C₁₋₈ alkyl, C₁₋₈ haloalkyl, —C₁₋₈alkoxy, —X¹—C₁₋₈ alkoxy, —OR^(a), —X¹—OR^(a), —NR^(a)R^(b),—X¹—NR^(a)R^(b), —S(O)₂R^(a), —S(O)₂NR^(a)R^(b), —X¹—S(O)₂R^(a), and—X¹—S(O)₂NR^(a)R^(b).

In some embodiments, each R² in Formulas I, I-1, I-2, Ia, Ia′, Ia1,Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb,IIb1, IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is independently selectedfrom the group consisting of halogen, —C₁₋₈ alkyl, C₁₋₈ haloalkyl, —C₁₋₈alkoxy, —X¹—C₁₋₈ alkoxy, —OR^(a), —X¹—OR^(a), —NR^(a)R^(b), and—X¹—NR^(a)R^(b).

In some embodiments, each R² in Formulas I, I-1, I-2, Ia, Ia′, Ia1,Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb,IIb1, IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is independently selectedfrom the group consisting of halogen, —C₁₋₈ alkyl, C₁₋₈ haloalkyl,—OR^(a), —X¹—OR^(a), —NR^(b)—C(O)—R^(2a), —NR^(a)R^(b), and—X¹—NR^(a)R^(b).

In some embodiments, each R² in Formulas I, I-1, I-2, Ia, Ia′, Ia1,Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb,IIb1, IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is independently selectedfrom the group consisting of —OR^(a), —X¹—OR^(a), —NR^(a)R^(b) or—X¹—NR^(a)R^(b).

In some embodiments, each R³ in Formulas I, I-1, I-2, Ia, Ia′ Ia1, Ia1′Ia2, Ia2′ Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′ IIb, IIc, III, IIIa,or IIIa′ is independently selected from the group consisting of halogen,—C₁₋₈ alkyl, —C₁₋₈ haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈ alkoxy,—C(O)—R^(3a), —SR^(a), —X¹—SR^(a), —OR^(a), —X¹—OR^(a), —NR^(a)R^(b),—X¹—NR^(a)R^(b), —S(O)₂R^(a), —S(O)₂NR^(a)R^(b), —X¹—S(O)₂R^(a), and—X¹—S(O)₂NR^(a)R^(b).

In some embodiments, each R³ in Formulas I, I-1, I-2, Ia, Ia′ Ia1, Ia1′Ia2, Ia2′ Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′ IIb, IIc, III, IIIa,or IIIa′ is independently selected from the group consisting of halogen,—C₁₋₈ alkyl, C₁₋₈ haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈ alkoxy, —OR^(a),—X¹—OR^(a)—NR^(a)R^(b), —X¹—NR^(a)R^(b), —S(O)₂R^(a), —S(O)₂NR^(a)R^(b),—X¹—S(O)₂R^(a), and —X¹—S(O)₂NR^(a)R^(b).

In some embodiments, each R³ in Formulas I, I-1, I-2, Ia, Ia′ Ia1, Ia1′Ia2, Ia2′ Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′ IIb, IIc, III, IIIa,or IIIa′ is independently selected from the group consisting of halogen,—C₁₋₈ alkyl, C₁₋₈ haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈ alkoxy, —OR^(a),—X¹—OR^(a), —NR^(a)R^(b), and —X¹—NR^(a)R^(b).

In some embodiments, each R³ in Formulas I, I-1, I-2, Ia, Ia′ Ia1, Ia1′Ia2, Ia2′ Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′ IIb, IIc, or III,IIIa, or IIIa′ is independently selected from the group consisting ofhalogen, —C₁₋₈ alkyl, C₁₋₈ haloalkyl, —OR^(a), —X¹—OR^(a), —NR^(a)R^(b),and —X¹—NR^(a)R^(b).

In some embodiments, each R³ in Formulas I, I-1, I-2, Ia, Ia′ Ia1, Ia1′Ia2, Ia2′ Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′ IIb, IIc, or III,IIIa, or IIIa′ is independently selected from the group consisting of—OR^(a), —X¹—OR^(a), —NR^(a)R^(b) or —X¹—NR^(a)R^(b).

In some embodiments, R^(1a) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is C₁₋₆ alkyl or C₁₋₆ haloalkyl.

In some embodiments, R^(1a) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is C₁₋₆ alkyl.

In some embodiments, R^(1a) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is C₂₋₆ alkyl or C₂₋₆ haloalkyl.

In some embodiments, R^(1a) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, IIc1, III, IIIa, IIIa′, or IIIa1 is C₂₋₆ alkyl.

In some embodiments, R^(1b) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, or IIc1 is —OR^(a).

In some embodiments, R^(1b) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, or IIc1 is —OH.

In some embodiments, R^(1b) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, or IIc1 is heterocycloalkyl wherein each heterocycloalkyl comprisesfrom 5 to 6 ring members having 1 to 3 heteroatom ring members selectedfrom the group consisting of nitrogen, oxygen, and sulfur, and isunsubstituted or substituted with one to four C₁₋₄ alkyl, —OH, and halo.

In some embodiments, R^(1b) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, or IIc1 is tetrahydropyran.

In some embodiments, R^(1b) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, or IIc1 is phenyl unsubstituted or substituted with one to fourC₁₋₄ alkyl, —OH, and halo.

In some embodiments, R^(1b) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, or IIc1 is 4-hydroxyphenyl.

In some embodiments, R^(1b) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′,Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1,IIc, or IIc is —NH₂ or —N(CH₃)₂.

In some embodiments, each R^(a) and R^(b) in Formulas I, I-1, I-2, Ia,Ia′, Ia1, Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′,IIa1, IIb, IIb1, IIc, or IIc1 is independently selected from the groupconsisting of H and C₁₋₂ alkyl.

In some embodiments, each X¹ in Formulas I, I-1, I-2, Ia, Ia′, Ia1,Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb,IIb1, IIc, or IIc1 is C₁₋₂ alkylene.

In some embodiments, each X¹ in Formulas I, I-1, I-2, Ia, Ia′, Ia1,Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1, IIb,IIb1, IIc, or IIc1 is C₁ alkylene.

In some embodiments, the subscript n in Formulas I, I-1, I-2, Ia, Ia′,Ia1, Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1,IIb, IIb1, IIc, or IIc1 is an integer from 1 to 3.

In some embodiments, the subscript n in Formulas I, I-1, I-2, Ia, Ia′,Ia1, Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1,IIb, IIb1, IIc, or IIc1 is 1.

In some embodiments, the subscript n in Formulas I, I-1, I-2, Ia, Ia′,Ia1, Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′, IIa1,IIb, IIb1, IIc, or IIc1 is 0.

In some embodiments, the subscript m in Formulas I, I-1, I-2, Ia, Ia′,Ia1, Ia1′ Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′ IIb, IIcis an integer from 1 to 2.

In some embodiments, the subscript m in Formulas I, I-1, I-2, Ia, Ia′,Ia1, Ia1′ Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′ IIb, IIcis 0.

In some embodiments, the subscript m in Formulas I, I-1, I-2, Ia, Ia′,Ia1, Ia1′ Ia2, Ia2′, Ib, Ib1, Ib2, Ic, Ic1, Ic2, II, IIa, IIa′ IIb, IIcis 1.

In some embodiments, R^(4a) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′Ia2, Ia2′ Ib, Ib1, Ib2, Ic, Ic1, or Ic2 is —OH or —NH₂.

In some embodiments, R^(4a) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′Ia2, Ia2′ Ib, Ib1, Ib2, Ic, Ic1, or Ic2 is —OH.

In some embodiments, R^(4a) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′Ia2, Ia2′ Ib, Ib1, Ib2, Ic, Ic1, or Ic2 is —O—C₁₋₄ alkyl.

In some embodiments, R^(4a) in Formulas I, I-1, I-2, Ia, Ia′, Ia1, Ia1′Ia2, Ia2′ Ib, Ib1, Ib2, Ic, Ic1, or Ic2 is —O—C(O)—C₁₋₄ alkyl.

In some embodiments, the compound of Formula I has the structure ofFormula II

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR¹ is selected from the group consisting of —NH—C(O)—R^(1a),—NH—C(O)—O—R^(1a); —NH—X¹—C(O)—R^(1a), and —NH—R^(1a);each R² and R³ is independently selected from the group consisting of—NH₂, —OH, —X¹—NH₂, —X¹—OH;R^(1a) is selected from the group consisting of C₂₋₆ alkyl; and C₁₋₆haloalkyl;each X¹ is C₁₋₂ alkylene;the subscript n is an integer from 0 to 2; andthe subscript m is 0 or 1.or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

In some embodiments, the compound of Formula IIa has the structure ofFormula IIa1

R¹ is selected from the group consisting of —NH—C(O)—R^(1a);R² is independently selected from the group consisting of —NH₂ or —OH;R^(1a) is selected from the group consisting of C₂₋₆ alkyl; and C₁₋₆haloalkyl; andthe subscript n is 0 or 1.

In some embodiments, the compound of Formula I has the structure ofFormula Ia

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR¹ is selected from the group consisting of —NH—C(O)—R^(1a);R² is independently selected from the group consisting of —NH₂ or —OH;R^(1a) is selected from the group consisting of C₂₋₆ alkyl; and C₁₋₆haloalkyl;R^(4a) is —OH;R^(4b) is H;the subscript n is 0 or 1; andthe subscript m is 0.

In some embodiments, the compound of Formula IIb has the structure ofFormula IIb1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein

R¹ is selected from the group consisting of —NH—C(O)—R^(1a);

R² is independently selected from the group consisting of —NH₂ or —OH;

R^(1a) is selected from the group consisting of C₂₋₆ alkyl; and C₁₋₆haloalkyl; and

the subscript n is 0 or 1.

In some embodiments, the compound of Formula IIc has the structure ofFormula IIc1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein

R¹ is selected from the group consisting of —NH—C(O)—R^(1a);

R² is independently selected from the group consisting of —NH₂ or —OH;

R^(1a) is selected from the group consisting of C₂₋₆ alkyl; and C₁₋₆haloalkyl; and

the subscript n is 0 or 1.

In some embodiments, the compound of Formula I is a selected from Table1.

TABLE 1 Particular Compounds Compound Structure 1.001

1.002

1.003

1.004

1.005

1.006

1.007

1.008

1.009

1.010

1.011

1.012

1.013

1.014

1.015

1.016

1.017

1.018

1.019

1.020

1.021

1.022

1.023

1.024

1.025

1.026

1.027

1.028

1.029

1.030

1.031

1.032

1.033

1.034

1.035

1.036

1.037

1.038

1.039

1.040

1.041

1.042

1.043

1.044

1.045

1.046

1.047

1.048

1.049

1.050

1.051

1.052

1.053

1.054

1.055

1.056

1.057

1.058

1.059

1.060

1.061

The cell culture media compositions for use in the methods of thepresent invention can include about 10-16,000 nM of the compound ofFormula I or a subembodiment disclosed herein, such as about 50-450 nM,100-400 nM, about 150-350 nM, about 200-300 nM, about 225-275 nM, orabout 240-260 nM, such as about 300-3000 nM, 500-2000 nM, about 550-1550nM, about 800-1200 nM, about 900-1100 nM, or about 950-1050 nM, or suchas any of about 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM,50 nM, 55 nM, 60 nM, 65 nM, 70 nM 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100nM, 105 nM, 110 nM, 115 nM, 120 nM, 125 nM, 130 nM, 135 nM, 140 nM, 145nM, 150 nM, 155 nM, 160 nM, 165 nM, 170 nM, 175 nM, 180 nM, 185 nM, 190nM, 195 nM, 200 nM, 205 nM, 210 nM, 215 nM, 220 nM, 225 nM, 230 nM, 240nM, 245 nM, 250 nM, 255 nM, 260 nM, 265 nM, 270 nM, 275 nM, 280 nM, 285nM, 290 nM, 295 nM, 300 nM, 325 nM, 350 nM, 400 nM, 425 nM, 450 nM, 475nM, 500 nM, 525 nM, 550 nM, 575 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700nM, 725 nM, 750 nM, 775 nM, 800 nM, 825 nM, 850 nM, 875 nM, 900 nM, 925nM, 950 nM, 975 nM, 1000 nM, 1100 nM, 1200 nM, 1300 nM, 1400 nM, 1500nM, 1600 nM, 1700 nM, 1800 nM, 1900 nM, 2000 nM, 2100 nM, 2200 nM, 2300nM, 2400 nM, 2500 nM, 2600 nM, 2700 nM, 2800 nM, 2900 nM, 3000 nM, 3100nM, 3200 nM, 3300 nM, 3400 nM, 3500 nM, 3600 nM, 3700 nM, 3800 nM, 3900nM, 4000 nM, 5000 nM, 6000 nM, 7000 nM, 8000 nM, 9000 nM, 10,000 nM,11,000 nM, 12,000 nM, 13,000 nM, 14,000 nM, 15,000 nM, 16,000 nM, ormore of the compound of Formula I or a subembodiment disclosed herein,including values falling in between these concentrations. In someembodiments, the culture media compositions for use in the methods ofthe present invention can include about 500 nM of the compound ofFormula I or a subembodiment disclosed herein. In some embodiments, theculture media compositions for use in the methods of the presentinvention can include about 800 nM of the compound of Formula I or asubembodiment disclosed herein. In some embodiments, the culture mediacompositions for use in the methods of the present invention can includeabout 1,600 nM of the compound of Formula I or a subembodiment disclosedherein. In some embodiments, the culture media compositions for use inthe methods of the present invention can include about 8,000 nM of thecompound of Formula I or a subembodiment disclosed herein.

Preparation of Compounds

Certain compounds of the invention can be prepared following methodologyas described in the Examples section of this document. In addition, thesyntheses of certain intermediate compounds that are useful in thepreparation of compounds of the invention are also described.

B. Cytokines and Growth Factors

The cell culture media (e.g. base media or feed media) for use in themethods disclosed herein can contain one or more cytokines or growthfactors. These agents promote the survival, maintenance, expansion, orenhancement of HSCs and can be procured via commercially availablesources.

Cell culture media for culturing HSCs can include thrombopoietin (TPO).Thrombopoietin is a glycoprotein hormone produced by the liver andkidney which regulates the production of platelets. It stimulates theproduction and differentiation of megakaryocytes, the bone marrow cellsthat bud off large numbers of platelets. The cell culture mediacompositions for use in the methods of the present invention can includeabout 50-250 ng/mL of TPO such as about 75-225 ng/mL, about 100-200ng/mL, or about 125-175 ng/mL, or such as any of about 75 ng/mL, 80ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, 100 ng/mL, 105 ng/mL, 110 ng/mL,115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 141ng/mL, 142 ng/mL, 143 ng/mL, 144 ng/mL, 145 ng/mL, 146 ng/mL, 147 ng/mL,148 ng/mL, 149 ng/mL, 150 ng/mL, 151 ng/mL, 152 ng/mL, 153 ng/mL, 154ng/mL, 155 ng/mL, 156 ng/mL, 157 ng/mL, 158 ng/mL, 159 ng/mL, 160 ng/mL,165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL,230 ng/mL, 235 ng/mL, 240 ng/mL, 245 ng/mL, or 250 ng/mL or more TPO,including values falling in between these concentrations. In someembodiments, the concentration of TPO in the media is about 150 ng/mL.

Any of the cell culture media disclosed herein can also include stemcell factor (also known as SCF, KIT-ligand, KL, or steel factor). SCF isa cytokine that binds to the c-KIT receptor (CD117) and which plays arole in the regulation of HSCs in bone marrow. SCF has been shown toincrease the survival of HSCs in vitro and contributes to theself-renewal and maintenance of HSCs in-vivo. The cell culture mediacompositions for use in the methods of the present invention can includeabout 5-100 ng/mL of SCF, such as about 10-90 ng/mL, about 20-80, ng/mLabout 30-70 ng/mL, about 40-60 ng/mL, or about 45-55 ng/mL, or such asany of about 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30 ng/mL,35 ng/mL, 40 ng/mL, 41 ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46ng/mL, 47 ng/mL, 48 ng/mL, 49 ng/mL, 50 ng/mL, 51 ng/mL, 52 ng/mL, 53ng/mL, 54 ng/mL, 55 ng/mL, 56 ng/mL, 57 ng/mL, 58 ng/mL, 59 ng/mL, 60ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95ng/mL, 100 ng/mL or more SCF, including values falling in between theseconcentrations. In some embodiments, the cell culture media compositionsfor use in the methods of the present invention can includeconcentrations at 100 ng/mL or above. Accordingly, concentrations of SCFalso include 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL 160 ng/mL, 165 ng/mL,170 ng/mL, 175 ng/mL, 180 ng/mL 185 ng/mL, 190 ng/mL, 200 ng/mL, or moreSCF, including values falling in between these concentrations. In someembodiments, the concentration of SCF in the media is about 100 ng/mL.

The cell culture media disclosed herein can also contain insulin-likegrowth factor 1 (IGF-1; also called somatomedin C). IGF-1 is a hormonesimilar in molecular structure to insulin. It plays an important role inchildhood growth and has anabolic effects in adults. The cell culturemedia compositions for use in the methods of the present invention caninclude about 100-400 ng/mL IGF-1, such as about 125-375 ng/mL, about150-350 ng/mL, about 175-325 ng/mL, about 200-300 ng/mL, about 225-275ng/mL, about 240-260 ng/mL, or about 245-255 ng/mL, or such as any ofabout 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL,130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL, 160ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL,195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242 ng/mL, 243 ng/mL,244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL, 249 ng/mL, 250ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255 ng/mL, 256 ng/mL,257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL, 270 ng/mL, 275ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300 ng/mL, 305 ng/mL,310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL, 335 ng/mL, 340ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365 ng/mL, 370 ng/mL,375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL, or 400 ng/mL ormore IGF-1, including values falling in between these concentrations. Insome embodiments, the concentration of IGF-1 is the media is about 250ng/mL

The cell culture media for culturing HSCs provided herein can furtherinclude fms-related tyrosine kinase 3 ligand (FLT3L). FLT3L is acytokine that stimulates cell growth, proliferation, anddifferentiation. The cell culture media compositions for use in themethods of the present invention can include about 20-400 ng/mL FLT3L,such as about 40-375 ng/mL, about 60-350 ng/mL, about 80-325 ng/mL,about 100-300 ng/mL, about 140-275 ng/mL, about 160-260 ng/mL, or about180-255 ng/mL, or such as any of about 20 ng/mL, 40 ng/mL, 60 ng/mL, 80ng/mL, 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL,130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL, 160ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL,195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242 ng/mL, 243 ng/mL,244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL, 249 ng/mL, 250ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255 ng/mL, 256 ng/mL,257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL, 270 ng/mL, 275ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300 ng/mL, 305 ng/mL,310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL, 335 ng/mL, 340ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365 ng/mL, 370 ng/mL,375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL, or 400 ng/mL ormore FLT3L, including values falling in between these concentrations. Insome embodiments, the concentration of FLT3L in the media is about 100ng/mL.

The cell culture media for culturing HSCs provided herein can furtherinclude human growth hormone (HGH). HGH is a protein hormone thatstimulates cell growth, proliferation, and differentiation. The cellculture media compositions for use in the methods of the presentinvention can include about 100-400 ng/mL EGF, such as about 125-375ng/mL, about 150-350 ng/mL, about 175-325 ng/mL, about 200-300 ng/mL,about 225-275 ng/mL, about 240-260 ng/mL, or about 245-255 ng/mL, orsuch as any of about 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL,155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL,220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL,249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL,270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL,335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL,or 400 ng/mL or more EGF, including values falling in between theseconcentrations. In some embodiments, the concentration of HGH in themedia is about 250 ng/mL.

The cell culture media for culturing HSCs provided herein can furtherinclude epidermal growth factor (EGF). EGF is a growth factor thatstimulates cell growth, proliferation, and differentiation by binding toits receptor EGFR. The cell culture media compositions for use in themethods of the present invention can include about 100-400 ng/mL EGF,such as about 125-375 ng/mL, about 150-350 ng/mL, about 175-325 ng/mL,about 200-300 ng/mL, about 225-275 ng/mL, about 240-260 ng/mL, or about245-255 ng/mL, or such as any of about 100 ng/mL, 105 ng/mL, 110 ng/mL,115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145ng/mL, 150 ng/mL, 155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL,180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL,241 ng/mL, 242 ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247ng/mL, 248 ng/mL, 249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL,254 ng/mL, 255 ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260ng/mL, 265 ng/mL, 270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL,295 ng/mL, 300 ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325ng/mL, 330 ng/mL, 335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL,360 ng/mL, 365 ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390ng/mL, 395 ng/mL, or 400 ng/mL or more EGF, including values falling inbetween these concentrations.

Any of the cell culture media disclosed herein can also includehepatocyte growth factor (HGF). HGF is a paracrine cellular growth,motility and morphogenic factor. It is secreted by mesenchymal cells andacts primarily upon epithelial cells and endothelial cells, but alsoacts on hematopoietic progenitor cells and T cells. HGF has been shownto have a major role in embryonic organ development, specifically inmyogenesis, in adult organ regeneration and in wound healing. The cellculture media compositions for use in the methods of the presentinvention can include about 100-400 ng/mL HGF, such as about 125-375ng/mL, about 150-350 ng/mL, about 175-325 ng/mL, about 200-300 ng/mL,about 225-275 ng/mL, about 240-260 ng/mL, or about 245-255 ng/mL, orsuch as any of about 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 120ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL,155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL,220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 241 ng/mL, 242ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247 ng/mL, 248 ng/mL,249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL, 254 ng/mL, 255ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260 ng/mL, 265 ng/mL,270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL, 295 ng/mL, 300ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325 ng/mL, 330 ng/mL,335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL, 360 ng/mL, 365ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390 ng/mL, 395 ng/mL,or 400 ng/mL or more HGF, including values falling in between theseconcentrations.

The cell culture media disclosed herein can also contain pleiotrophin(PTN). PTN is a developmentally regulated protein that has been shown tobe involved in tumor growth and metastasis presumably by activatingtumor angiogenesis. The cell culture media compositions for use in themethods of the present invention can include about 100-400 ng/mL PTN,such as about 125-375 ng/mL, about 150-350 ng/mL, about 175-325 ng/mL,about 200-300 ng/mL, about 225-275 ng/mL, about 240-260 ng/mL, or about245-255 ng/mL, or such as any of about 100 ng/mL, 105 ng/mL, 110 ng/mL,115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL, 135 ng/mL, 140 ng/mL, 145ng/mL, 150 ng/mL, 155 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL,180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL,241 ng/mL, 242 ng/mL, 243 ng/mL, 244 ng/mL, 245 ng/mL, 246 ng/mL, 247ng/mL, 248 ng/mL, 249 ng/mL, 250 ng/mL, 251 ng/mL, 252 ng/mL, 253 ng/mL,254 ng/mL, 255 ng/mL, 256 ng/mL, 257 ng/mL, 258 ng/mL, 259 ng/mL, 260ng/mL, 265 ng/mL, 270 ng/mL, 275 ng/mL, 280 ng/mL, 285 ng/mL, 290 ng/mL,295 ng/mL, 300 ng/mL, 305 ng/mL, 310 ng/mL, 315 ng/mL, 320 ng/mL, 325ng/mL, 330 ng/mL, 335 ng/mL, 340 ng/mL, 345 ng/mL, 350 ng/mL, 355 ng/mL,360 ng/mL, 365 ng/mL, 370 ng/mL, 375 ng/mL, 380 ng/mL, 385 ng/mL, 390ng/mL, 395 ng/mL, or 400 ng/mL or more PTN, including values falling inbetween these concentrations. In some embodiments, PTN does not improvethe maintenance or enhancement of hematopoietic stem cells.

In further embodiments, the cell culture media compositions disclosedherein can additionally contain basic fibroblast growth factor (bFGF,FGF2 or FGF-β). bFGF is a critical component of human embryonic stemcell culture medium. However, while the growth factor is necessary forthe cells to remain in an undifferentiated state, the mechanisms bywhich it does this are poorly defined. The cell culture mediacompositions for use in the methods of the present invention can includeabout 25-225 ng/mL of bFGF such as about 50-200 ng/mL, about 100-200ng/mL, about 100-150 ng/mL, or about 115-135 ng/mL, or such as any ofabout 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95 ng/mL, 100 ng/mL, 105ng/mL, 110 ng/mL, 115 ng/mL, 116 ng/mL, 117 ng/mL, 118 ng/mL, 119 ng/mL,120 ng/mL, 121 ng/mL, 122 ng/mL, 123 ng/mL, 124 ng/mL, 125 ng/mL, 126ng/mL, 127 ng/mL, 128 ng/mL, 129 ng/mL, 130 ng/mL, 131 ng/mL, 132 ng/mL,133 ng/mL, 134 ng/mL, 135 ng/mL, 140 ng/mL, 141 ng/mL, 142 ng/mL, 143ng/mL, 144 ng/mL, 145 ng/mL, 146 ng/mL, 147 ng/mL, 148 ng/mL, 149 ng/mL,150 ng/mL, 151 ng/mL, 152 ng/mL, 153 ng/mL, 154 ng/mL, 155 ng/mL, 156ng/mL, 157 ng/mL, 158 ng/mL, 159 ng/mL, 160 ng/mL, 165 ng/mL, 170 ng/mL,175 ng/mL, 180 ng/mL, 185 ng/mL, 190 ng/mL, 195 ng/mL, 200 ng/mL, 205ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL, 225 ng/mL, 230 ng/mL, 235 ng/mL,240 ng/mL, 245 ng/mL, or 250 ng/mL or more bFGF, including valuesfalling in between these concentrations.

Any of the cell culture media disclosed herein can also includeangiopoietin 1 (ANG1). ANG1 is a member of the angiopoietin family ofvascular growth factors that play a role in embryonic and postnatalangiogenesis. The cell culture media compositions for use in the methodsof the present invention can include about 25-225 ng/mL of ANG1 such asabout 50-200 ng/mL, about 100-200 ng/mL, about 100-150 ng/mL, or about115-135 ng/mL, or such as any of about 75 ng/mL, 80 ng/mL, 85 ng/mL, 90ng/mL, 95 ng/mL, 100 ng/mL, 105 ng/mL, 110 ng/mL, 115 ng/mL, 116 ng/mL,117 ng/mL, 118 ng/mL, 119 ng/mL, 120 ng/mL, 121 ng/mL, 122 ng/mL, 123ng/mL, 124 ng/mL, 125 ng/mL, 126 ng/mL, 127 ng/mL, 128 ng/mL, 129 ng/mL,130 ng/mL, 131 ng/mL, 132 ng/mL, 133 ng/mL, 134 ng/mL, 135 ng/mL, 140ng/mL, 141 ng/mL, 142 ng/mL, 143 ng/mL, 144 ng/mL, 145 ng/mL, 146 ng/mL,147 ng/mL, 148 ng/mL, 149 ng/mL, 150 ng/mL, 151 ng/mL, 152 ng/mL, 153ng/mL, 154 ng/mL, 155 ng/mL, 156 ng/mL, 157 ng/mL, 158 ng/mL, 159 ng/mL,160 ng/mL, 165 ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL, 185 ng/mL, 190ng/mL, 195 ng/mL, 200 ng/mL, 205 ng/mL, 210 ng/mL, 215 ng/mL, 220 ng/mL,225 ng/mL, 230 ng/mL, 235 ng/mL, 240 ng/mL, 245 ng/mL, or 250 ng/mL ormore ANG1, including values falling in between these concentrations.

Interleukin 10 (IL-10) can also be a component of any of the cellculture media compositions disclosed herein. IL-10 is a cytokine withmultiple, pleiotropic, effects in immunoregulation and inflammation. Itdownregulates the expression of Th1 cytokines, MHC class II antigens,and co-stimulatory molecules on macrophages. It also enhances B cellsurvival, proliferation, and antibody production. IL-10 can block NF-κBactivity, and is involved in the regulation of the JAK-STAT signalingpathway. The cell culture media compositions for use in the methods ofthe present invention can include about 1-25 ng/mL of IL-10 such asabout 5-20 ng/mL, 10-20 ng/mL, or 12-18 ng/mL, such as any of about 1ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9ng/mL, 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23ng/mL, 24 ng/mL, or 25 ng/mL of IL-10.

Interleukin 3 (IL-3) can also be a component of any of the cell culturemedia compositions disclosed herein. IL-3 is a cytokine with multiple,pleiotropic, effects in immunoregulation and inflammation. The cellculture media compositions for use in the methods of the presentinvention can include about 1-25 ng/mL of IL-3 such as about 5-20 ng/mL,10-20 ng/mL, or 12-18 ng/mL, such as any of about 1 ng/mL, 2 ng/mL, 3ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL,11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, or 25ng/mL of IL-3. In some embodiments, the cell culture media compositionsfor use in the methods of the present invention can includeconcentrations at 25 ng/mL or above. Accordingly, concentrations of IL-3also include 10-140 ng/mL, about 30-130, ng/mL about 50-120 ng/mL, about70-110 ng/mL, or about 95-105 ng/mL, or such as any of about 30 ng/mL,35 ng/mL, 40 ng/mL, 41 ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46ng/mL, 47 ng/mL, 48 ng/mL, 49 ng/mL, 50 ng/mL, 51 ng/mL, 52 ng/mL, 53ng/mL, 54 ng/mL, 55 ng/mL, 56 ng/mL, 57 ng/mL, 58 ng/mL, 59 ng/mL, 60ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95ng/mL, 100 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL,135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL 160 ng/mL, 165ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL 185 ng/mL, 190 ng/mL, 200 ng/mL,or more IL-3, including values falling in between these concentrations.In some embodiments, the concentration of IL-3 in the media is about 100ng/mL.

Interleukin 6 (IL-6) can also be a component of any of the cell culturemedia compositions disclosed herein. IL-6 is a cytokine with multiple,pleiotropic, effects in immunoregulation and inflammation. The cellculture media compositions for use in the methods of the presentinvention can include about 1-25 ng/mL of IL-6 such as about 5-20 ng/mL,10-20 ng/mL, or 12-18 ng/mL, such as any of about 1 ng/mL, 2 ng/mL, 3ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL,11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, or 25ng/mL of IL-6. In some embodiments, the cell culture media compositionsfor use in the methods of the present invention can includeconcentrations at 25 ng/mL or above. Accordingly, concentrations of IL-6also include 10-140 ng/mL, about 30-130, ng/mL about 50-120 ng/mL, about70-110 ng/mL, or about 95-105 ng/mL, or such as any of about 30 ng/mL,35 ng/mL, 40 ng/mL, 41 ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45 ng/mL, 46ng/mL, 47 ng/mL, 48 ng/mL, 49 ng/mL, 50 ng/mL, 51 ng/mL, 52 ng/mL, 53ng/mL, 54 ng/mL, 55 ng/mL, 56 ng/mL, 57 ng/mL, 58 ng/mL, 59 ng/mL, 60ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90 ng/mL, 95ng/mL, 100 ng/mL, 110 ng/mL, 115 ng/mL, 120 ng/mL, 125 ng/mL, 130 ng/mL,135 ng/mL, 140 ng/mL, 145 ng/mL, 150 ng/mL, 155 ng/mL 160 ng/mL, 165ng/mL, 170 ng/mL, 175 ng/mL, 180 ng/mL 185 ng/mL, 190 ng/mL, 200 ng/mL,or more IL-6, including values falling in between these concentrations.In some embodiments, the concentration of IL-6 in the media is about 100ng/mL.

The cell culture media disclosed herein can also contain vascularendothelial growth factor 165 (VEGF165), which belongs to the PDGF/VEGFgrowth factor family. Many cell types secrete VEGF165, which it is apotent angiogenic factor and mitogen that stimulates proliferation,migration, and formation of endothelial cells. The cell culture mediacompositions for use in the methods of the present invention can includeabout 5-100 ng/mL of VEGF165, such as about 10-90 ng/mL, about 20-80,ng/mL about 30-70 ng/mL, about 40-60 ng/mL, or about 45-55 ng/mL, orsuch as any of about 5 ng/mL, 10 ng/mL, 15 ng/mL, 20 ng/mL, 25 ng/mL, 30ng/mL, 35 ng/mL, 40 ng/mL, 41 ng/mL, 42 ng/mL, 43 ng/mL, 44 ng/mL, 45ng/mL, 46 ng/mL, 47 ng/mL, 48 ng/mL, 49 ng/mL, 50 ng/mL, 51 ng/mL, 52ng/mL, 53 ng/mL, 54 ng/mL, 55 ng/mL, 56 ng/mL, 57 ng/mL, 58 ng/mL, 59ng/mL, 60 ng/mL, 65 ng/mL, 70 ng/mL, 75 ng/mL, 80 ng/mL, 85 ng/mL, 90ng/mL, 95 ng/mL, 100 ng/mL or more VEGF165, including values falling inbetween these concentrations.

The cell culture media disclosed herein can also contain vascularendothelial growth factor C (VEGF-C), which belongs to the PDGF/VEGFgrowth factor family. Many cell types secrete VEGF-C, which functions inangiogenesis, and endothelial cell growth, stimulating proliferation andmigration and also has effects on the permeability of blood vessels. Thecell culture media compositions for use in the methods of the presentinvention can include about 50-1000 ng/mL of VEGF-C, such as about100-900 ng/mL, about 200-800, ng/mL about 300-700 ng/mL, about 400-600ng/mL, or about 450-550 ng/mL, or such as any of about 50 ng/mL, 100ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 350 ng/mL, 400 ng/mL,410 ng/mL, 420 ng/mL, 430 ng/mL, 440 ng/mL, 450 ng/mL, 460 ng/mL, 470ng/mL, 480 ng/mL, 490 ng/mL, 500 ng/mL, 510 ng/mL, 520 ng/mL, 530 ng/mL,540 ng/mL, 550 ng/mL, 560 ng/mL, 570 ng/mL, 580 ng/mL, 590 ng/mL, 600ng/mL, 650 ng/mL, 700 ng/mL, 750 ng/mL, 800 ng/mL, 850 ng/mL, 900 ng/mL,950 ng/mL, 1000 ng/mL or more VEGF-C, including values falling inbetween these concentrations.

In yet additional embodiments, the cell culture media compositionsdisclosed herein can contain laminins, which are high-molecular weight(˜400 kDa) proteins of the extracellular matrix. They are a majorcomponent of the basal lamina (one of the layers of the basementmembrane), a protein network foundation for most cells and organs. Thelaminins are an important and biologically active part of the basallamina, influencing cell differentiation, migration, and adhesion. Thecell culture media compositions for use in the methods of the presentinvention can include about 500-1000 ng/mL laminin, such as about600-900 ng/mL, about 700-800 ng/mL, about 725-775 ng/mL, or about745-755 ng/mL, or such as any of about 500 ng/mL, 525 ng/mL, 550 ng/mL,575 ng/mL, 600 ng/mL, 625 ng/mL, 650 ng/mL, 675 ng/mL, 700 ng/mL, 705ng/mL, 710 ng/mL, 715 ng/mL, 720 ng/mL, 725 ng/mL, 730 ng/mL, 735 ng/mL,740 ng/mL, 741 ng/mL, 742 ng/mL, 743 ng/mL, 744 ng/mL, 745 ng/mL, 746ng/mL, 747 ng/mL, 748 ng/mL, 749 ng/mL, 750 ng/mL, 751 ng/mL, 752 ng/mL,753 ng/mL, 754 ng/mL, 755 ng/mL, 756 ng/mL, 757 ng/mL, 758 ng/mL, 759ng/mL, 760 ng/mL, 765 ng/mL, 770 ng/mL, 775 ng/mL, 780 ng/mL, 785 ng/mL,790 ng/mL, 795 ng/mL, 800 ng/mL, 825 ng/mL, 850 ng/mL, 875 ng/mL, 900ng/mL, 925 ng/mL, 950 ng/mL, 975 ng/mL, 1000 ng/mL or more laminin,including values falling in between these concentrations.

C. Other Small Molecules

The cell culture media for use in the methods disclosed herein canadditionally contain various small molecule inhibitors, such as caspaseinhibitors, DNA methylation inhibitors, p38 MAPK inhibitors, glycogensynthase kinase 3 (GSK3) inhibitors, and/or JAK/STAT inhibitors. In oneembodiment, the DMSO concentration of the cell culture media does notexceed 0.025% v/v.

In some embodiments, the cell culture media for use in the methodsdisclosed herein includes one or more caspase inhibitors. Caspases are afamily of cysteine proteases that play essential roles in apoptosis(programmed cell death), necrosis, and inflammation. As of November2009, twelve caspases have been identified in humans. There are twotypes of apoptotic caspases: initiator (apical) caspases and effector(executioner) caspases. Initiator caspases (e.g., CASP2, CASP8, CASP9,and CASP10) cleave inactive pro-forms of effector caspases, therebyactivating them. Effector caspases (e.g., CASP3, CASP6, CASP7) in turncleave other protein substrates within the cell, to trigger theapoptotic process. The cell culture media compositions for use in themethods of the present invention can include about 1-10 μg/mL caspaseinhibitor, such as any of about 2-8 μg/mL, about 3-7 μg/mL, or about 4-6μg/mL, or such as any of about 1 μg/mL, 2 μg/mL, 3 μg/mL, 4 μg/mL, 5μg/mL, 6 μg/mL, 7 μg/mL, 8 μg/mL, 9 μg/mL, 10 μg/mL or more caspaseinhibitor. In one embodiment, the caspase inhibitor is Z-VAD-FMK.

The cell culture media for use in the methods disclosed herein caninclude one or more DNA methylation inhibitors. DNA methylation is aprocess by which methyl groups are added to DNA which modifies itsfunction. When located in a gene promoter, DNA methylation typicallyacts to repress gene transcription. The cell culture media compositionsfor use in the methods of the present invention can include about300-700 nM DNA methylation inhibitors, such as about 350-650 nM, about400-600 nM, about 450-550 nM, about 475-525 nM, or about 490-510 nM orsuch as any of about 300 nM, 325 nM, 350 nM, 400 nM, 425 nM, 430 nM, 435nM, 440 nM, 445 nM, 450 nM, 455 nM, 460 nM, 465 nM, 470 nM, 475 nM, 480nM, 485 nM, 490 nM, 491 nM, 492 nM, 493 nM, 494 nM, 495 nM, 496 nM, 497nM, 498 nM, 499 nM, 500 nM, 501 nM, 502 nM, 503 nM, 504 nM, 505 nM, 506nM, 507 nM, 508 nM, 509 nM, 510 nM, 515 nM, 520 nM, 525 nM, 530 nM, 535nM, 540 nM, 545 nM, 550 nM, 555 nM, 560 nM, 565 nM, 570 nM, 575 nM, 600nM, 625 nM, 650 nM, 675 nM, 700 nM, or more DNA methylation inhibitors,including values falling in between these concentrations. In someembodiments, the DNA methylation inhibitor is epigallocatechin gallate(EGCG). In other embodiments, the cell culture media compositions foruse in the methods of the present invention can include about 0.25-3 μMDNA methylation inhibitors, such as about 0.5-2.5 μM, about 1-2 μM, orabout 1.25-1.75 μM, such as any of about 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5μM, or 3 μM or more DNA methylation inhibitors, including values fallingin between these concentrations. In some embodiments, the DNAmethylation inhibitor is Oct4-activating compound 1 (OAC1).

Any of the cell culture media disclosed herein can also include a p38MAPK inhibitor. p38 mitogen-activated protein kinases are a class ofmitogen-activated protein kinases that are responsive to stress stimuli,such as cytokines, ultraviolet irradiation, heat shock, and osmoticshock, and are involved in cell differentiation, apoptosis andautophagy. The cell culture media compositions for use in the methods ofthe present invention can include about 400-800 nM p38 MAPK inhibitor,such as about 500-700 nM, about 550-650 nM, about 600-650 nM, or about615-635 nM, or such as any of about 400 nM, 425 nM, 450 nM, 475 nM, 500nM, 525 nM, 550 nM, 575 nM, 600 nM, 605 nM, 610 nM, 615 nM, 616 nM, 617nM, 618 nM, 619 nM, 620 nM, 621 nM, 622 nM, 623 nM, 624 nM, 625 nM, 626nM, 627 nM, 628 nM, 629 nM, 630 nM, 631 nM, 632 nM, 633 nM, 634 nM, 635nM, 640 nM, 645 nM, 650 nM, 655 nM, 660 nM, 665 nM, 670 nM, 675 nM, 680nM, 685 nM, 690 nM, 695 nM, 700 nM, 725 nM, 750 nM, 775 nM, 800 nM, ormore p38 MAPK inhibitor, including values falling in between theseconcentrations. In some embodiments, the p38 MAPK inhibitor is BIRB796.

In yet additional embodiments, the cell culture media compositionsdisclosed herein can contain a glycogen synthase kinase 3 (GSK3)inhibitor. GSK3 is a serine/threonine protein kinase that mediates theaddition of phosphate molecules onto serine and threonine amino acidresidues. Phosphorylation of a protein by GSK-3 usually inhibits theactivity of its downstream target. GSK-3 is active in a number ofcentral intracellular signaling pathways, including cellularproliferation, migration, glucose regulation, and apoptosis. The cellculture media compositions for use in the methods of the presentinvention can include about 0.25-2 μM GSK3 inhibitor, such as about0.5-1.5 μM, or 1.75-1.25 μM, such as about 0.25 μM, 0.3 μM, 0.4 μM, 0.5μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 1.1 μM, 1.2 μM, 1.3 μM, 1.4μM, 1.5 μM, 1.6 μM, 1.7 μM, 1.8 μM, 1.9 μM, or 2 μM or more GSK3inhibitor, including values falling in between these concentrations. Insome embodiments, the GSK3 inhibitor is CHIR99021.

In further embodiments, the cell culture media compositions disclosedherein can additionally contain a retinoic acid receptor (RAR)antagonist or the media can include a controlled or reduced amount ofretinoic acid to restric retinoic acid signaling. The RAR is a nuclearreceptor as well as a transcription factor that is activated by bothall-trans retinoic acid and 9-cis retinoic acid. In some embodimentsretinoic acid signaling is reduced by limiting the amount of retinoicacid in the media.

In further embodiments, the cell culture media compositions disclosedherein can additionally contain a retinoic acid receptor (RAR)antagonist. The cell culture media compositions for use in the methodsof the present invention can include about 10-300 nM RAR antagonist,such as about 25-175 nM, about 50-150, or about 75-125, or such as anyof about 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM,55 nM, 60 nM, 65 nM, 70 nM 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM,105 nM, 110 nM, 115 nM, 120 nM, 125 nM, 130 nM, 135 nM, 140 nM, 145 nM,150 nM, 155 nM, 160 nM, 165 nM, 170 nM, 175 nM, 180 nM, 185 nM, 190 nM,191 nM, 192 nM, 193 nM, 194 nM, 195 nM, 196 nM, 197 nM, 198 nM, 199 nM,200 nM, 201 nM, 202 nM, 203 nM, 204 nM, 205 nM, 206 nM, 207 nM, 208 nM,209 nM, 210 nM, 215 nM, 220 nM, 225 nM, 230 nM, 235 nM, 240 nM, 241 nM,242 nM, 243 nM, 244 nM, 245 nM, 246 nM, 247 nM, 248 nM, 249 nM, 250 nM,251 nM, 252 nM, 253 nM, 254 nM, 255 nM, 256 nM, 257 nM, 258 nM, 259 nM,260 nM, 265 nM, 270 nM, 275 nM, 280 nM, 285 nM, 290 nM, 295 nM, 300 nMor more RAR antagonist, including values falling in between theseconcentrations. In some embodiments, the RAR antagonist is ER50891. Insome embodiments, the concentration of ER50891 is about 100 nM.

The cell culture media disclosed herein can also include a JAK/STATinhibitor. The JAK-STAT signaling pathway transmits information fromextracellular chemical signals to the nucleus resulting in DNAtranscription and expression of genes involved in immunity,proliferation, differentiation, apoptosis and oncogenesis. The cellculture media compositions for use in the methods of the presentinvention can include about 300-700 nM JAK/STAT inhibitor, such as about350-650 nM, about 400-600 nM, about 450-550 nM, about 475-525 nM, orabout 490-510 nM or such as any of about 300 nM, 325 nM, 350 nM, 400 nM,425 nM, 430 nM, 435 nM, 440 nM, 445 nM, 450 nM, 455 nM, 460 nM, 465 nM,470 nM, 475 nM, 480 nM, 485 nM, 490 nM, 491 nM, 492 nM, 493 nM, 494 nM,495 nM, 496 nM, 497 nM, 498 nM, 499 nM, 500 nM, 501 nM, 502 nM, 503 nM,504 nM, 505 nM, 506 nM, 507 nM, 508 nM, 509 nM, 510 nM, 515 nM, 520 nM,525 nM, 530 nM, 535 nM, 540 nM, 545 nM, 550 nM, 555 nM, 560 nM, 565 nM,570 nM, 575 nM, 600 nM, 625 nM, 650 nM, 675 nM, 700 nM, or more JAK/STATinhibitor, including values falling in between these concentrations. Insome embodiments, the JAK/STAT inhibitor is Tofacitinib.

In addition to the inhibitor molecules described above, any of the cellculture media compositions disclosed herein can also contain fetalbovine serum (FBS) in concentrations ranging from 1-20% v/v, such asabout 2-18% v/v, about 5-15% v/v, about 7.5-12.5% v/v or such as any ofabout 1% v/v, 2% v/v, 3% v/v, 4% v/v, 5% v/v, 6% v/v, 7% v/v, 8% v/v, 9%v/v, 10% v/v, 11% v/v, 12% v/v, 13% v/v, 14% v/v, 15% v/v, 16% v/v, 17%v/v, 18% v/v, 19% v/v, or 20% v/v or more FBS, including values fallingin between these percentages. In some embodiments, the FBS is heatinactivated FBS. In some embodiments, the concentration of FBS in themedium is about 10% v/v.

In addition to the inhibitor molecules described above, any of the cellculture media compositions disclosed herein can also contain addedsalts, for example KCl, NaCl, MgCl, or CaCl₂. In one example, CaCl₂ maybe added to achieve concentrations ranging from 300-380 mOsm, such asabout 300 mOsm, about 310 mOsm, about 320 mOsm, about 330 mOsm, about340 mOsm, about 350 mOsm, about 360 mOsm, about 370 mOsm, about 380mOsm, or more CaCl₂, including values falling in between these numbers.High osmolarity CaCl₂ may also be used to select against non-multipotentcells, selecting for an HSC phenotype.

In addition to the inhibitor molecules described above, any of the cellculture media compositions disclosed herein may be adjusted to comprisean overall higher osmolarity. Multipotent stem cells may be betteradapted to withstand atypical osmolarity (e.g., a high osmolarity mediamay select against non-stem cell phenotypes.) Osmolarity may beadjusted, for example, by the addition of salts as above, or by glucose.

IV. Methods of the Invention

A. Maintaining and/or Enhancing the Expansion of Hematopoietic StemCells in Culture

Provided herein are methods for maintaining and/or enhancing theexpansion of hematopoietic stem cells (HSCs) in culture. The methodinvolves contacting a source of CD34+ cells in culture with a compoundof Formula I, I-1, I-2, Ia, Ia′, Ia1, Ia1′, Ia2, Ia2′, Ib, Ib1, Ib2, Ic,Ic1, Ic2, II, IIa, IIa′, IIa1, IIb, IIb1, IIc, IIc1, III, IIIa, IIIa′,or IIIa1 or a compound of Table 1. In some embodiments, the methodsprovided herein do not include a tetraspanin. In some embodiments, themethods provided herein also include a retinoic acid receptor (RAR)inhibitor or modulator. In some embodiments, the RAR inhibitors isER50891.

1. Sources of CD34+ Cells

The methods of the present invention require a source of CD34+ bloodcells, or in some examples CD34low/−, CD133+ cells. These cells can beobtained from tissue sources such as, e.g., bone marrow, cord blood,placental blood, mobilized peripheral blood, non-mobilized peripheralblood, or the like, or combinations thereof.

In some embodiments, hematopioetic stem cells and/or progenitors arederived from one or more sources of CD34+ cells. CD34+ cells can, incertain embodiments, express or lack the cellular marker CD133. Thus, inspecific embodiments, the hematopoietic cells useful in the methodsdisclosed herein are CD34+CD133+ or CD34+CD133−. In other embodiments,CD34+ cells can express or lack the cellular marker CD90. As such, inthese embodiments, the hematopoietic cells useful in the methodsdisclosed herein are CD34+CD90+ or CD34+CD90−. Thus, populations ofCD34+ cells, or in some examples CD34low/−, CD133+ cells, can beselected for use in the methods disclosed herein on the basis of thepresence of markers that indicate an undifferentiated state, or on thebasis of the absence of lineage markers indicating that at least somelineage differentiation has taken place.

CD34+ cells used in the methods provided herein can be obtained from asingle individual, e.g., from a source of non-mobilized peripheralblood, or from a plurality of individuals, e.g., can be pooled. In someembodiments, the CD34+ cells from a single individual are sourced fromnon-mobilized peripheral blood, mobilized peripheral blood, placentalblood, or umbilical cord blood, Where the CD34+ cells are obtained froma plurality of individuals and pooled, it is preferred that thehematopoietic cells be obtained from the same tissue source. Thus, invarious embodiments, the pooled hematopoietic cells are all from, forexample, placenta, umbilical cord blood, peripheral blood (mobilized ornon-mobilized), and the like.

Interestingly, cells enhanced and expanded by methods of the presentinvention are, for example, phenotypically similar to cord blood.Accordingly, it may be possible to use cells expanded and enhanced bymethods described herein as a source for further expansion andenhancement. For example, it may be possible, following an initialexpansion and enhancement to allow, or gently encourage, cells towarddifferentiation. These cells may be allowed to expand and can then bebrought back from a differentiated, or near differentiated state, byfollowing the methods of the invention utilized in the initialexpansion/enhancement step. This expansion of differentiated, or nearlydifferentiated cells which can then be returned to a multipotent statemay occur over multiple cycles.

CD34+ cells, or in some examples CD34low/−, CD133+ cells, can beisolated from a source using any conventional means known in the artsuch as, without limitation, positive selection against stem cellmarkers, negative selection against lineage markers, size exclusion,detection of metabolic differences in the cells, detection ofdifferences in clearance or accumulation of a substance by the cell,adhesion differences, direct culturing of buffy coat under conditionsexclusively supportive for stem cells. The source of CD34+ cells for usein the methods of the present invention can contain a number ofsub-species of hematopoietic progenitor cells including, withoutlimitation, one or more of CD34+ hematopoietic progenitors; CD34+ earlyhematopoietic progenitors and/or stem cells; CD133+ early hematopoieticprogenitors and/or stem cells; CD90+ early hematopoietic progenitorsand/or stem cells; CD45RA− early hematopoietic progenitors and/or stemcells; and/or CD38 low/− early hematopoietic progenitors and/or stemcells.

2. Maintaining HSCs in Culture

CD34+ cells derived from the sources described above are cultured in anyof the cell culture media described herein. These media maintain andenhance the hematopoietic stem cell phenotype. Furthermore, the additionof a compound of Formula I or a subembodiment disclosed therein augmentsthese effects. Specifically, use of a compound of Formula I or asubembodiment described herein in the culture media increases the rateof expansion of HSCs while maintaining (and usually improving) allmeasured stem cell markers (such as, but not limited to CD133 and CD90positive cells). These improvements can be seen after as little as 3days of culture. In some embodiments, the media provided herein does notinclude a tetraspanin. In some embodiments, media provided herein alsoincludes a retinoic acid receptor (RAR) inhibitor or modulator. In someembodiments, the RAR inhibitor is ER50891.

In particular, source cells cultured in any of the cell culture mediadescribed herein exhibit increased numbers of CD133+ and/or CD90+positive cells compared to source cells that are not cultured in any ofthe media described herein after about any of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 days or more inculture. Specifically, source cells cultured in the media describedherein using the methods disclosed herein exhibited around 1.5, 1.6,1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5,4, 4.5, 5, 7.5, or 10 or more times the number of CD133+ and/or CD90+positive cells compared to source cells that are not cultured in any ofthe media described herein after about any of 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 days or more inculture.

Source cells cultured in the cell culture media described herein alsoexhibit increased number of CD90+/CD38 low/− cells compared to sourcecells that are not cultured in any of the media described herein afterabout any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30,35, 40, 45, or 50 days or more in culture. Specifically, source cellscultured in the media described herein using the methods disclosedherein exhibited around 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4,2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 7.5, 10, 12.5, 15, 17.5, or20 or more times the number of CD90+/CD38 low/− cells compared to sourcecells that are not cultured in any of the media described herein afterabout any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30,35, 40, 45, or 50 days or more in culture.

The cell culture methods disclosed herein include culturing cells underlow oxygen conditions. As used herein, the phrase “low oxygenconditions” refers to an atmosphere to which the cultured cells areexposed having less than about 10% oxygen, such as any of about 10%,9.5, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, or 5%, 4.5%, 4%, 3.5%, 3%,2.75%, 2.5%, 2.25%, 2%, 1.75%, 1.5%, 1.25%, 1%, 0.9%, 0.8%, 0.7%, 0.6%,or 0.5% or less oxygen. “Low oxygen conditions” can also refer to anyrange in between 0.5% and 10% oxygen. Control of atmospheric oxygen incell culture can be performed by any means known in the art, such as byaddition of nitrogen.

The cell culture methods disclosed herein include culturing cells underatmospheric oxygen conditions. As used herein, the phrase “atmosphericoxygen conditions” refers to an atmosphere including about 20% oxygen.

The invention also contemplates populations of cells that are made bythe methods described herein. Populations of cells containing HSCsprovided herein confer the advantages found in cord blood. A person ofskill in the art would readily recognize the characteristics of stemcells from cord blood and the advantageous properties therein. In someembodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% of the populations of cells containing HSCs provided herein areexpanded HSC cells. In some embodiments, the expanded HSC cells in thepopulations of cells have retained their stem cell phenotype for anextended period of time. For example, in some embodiments, populationsof cells containing HSCs include expanded HSC cells with cell surfacephenotypes that include CD45+, CD34+, CD133+, CD90+, CD45RA-, and/orCD38 low/− and have been cultured in vitro for at least 3, 7, 10, 13,14, 20, 25, 30, 40, or 50 or more days. In some embodiments, populationsof cells containing HSCs include expanded HSC cells with cell surfacephenotypes that includes CD133+ and/or CD90+ and have been cultured invitro for at least 3, 7, 10, 13, 14 or more days.

B. Methods of Treatment

Provided herein are methods for treating an individual in need ofhematopoietic reconstitution. The method involves administering to theindividual a therapeutic agent containing any of the cultured HSCsderived according to the methods of the present invention.

One of ordinary skill in the art may readily determine the appropriateconcentration, or dose of the cultured HSCs disclosed herein fortherapeutic administration. The ordinary artisan will recognize that apreferred dose is one that produces a therapeutic effect, such aspreventing, treating and/or reducing diseases, disorders and injuries,in a patient in need thereof. Of course, proper doses of the cells willrequire empirical determination at time of use based on severalvariables including but not limited to the severity and type of disease,injury, disorder or condition being treated; patient age, weight, sex,health; other medications and treatments being administered to thepatient; and the like.

An effective amount of cells may be administered in one dose, but is notrestricted to one dose. Thus, the administration can be two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, ormore, administrations of pharmaceutical composition. Where there is morethan one administration of a therapeutic agent in the present methods,the administrations can be spaced by time intervals of one minute, twominutes, three, four, five, six, seven, eight, nine, ten, or moreminutes, by intervals of about one hour, two hours, three, four, five,six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24 hours, and so on. In the context of hours, the term“about” means plus or minus any time interval within 30 minutes. Theadministrations can also be spaced by time intervals of one day, twodays, three days, four days, five days, six days, seven days, eightdays, nine days, ten days, 11 days, 12 days, 13 days, 14 days, 15 days,16 days, 17 days, 18 days, 19 days, 20 days, 21 days, and combinationsthereof. The invention is not limited to dosing intervals that arespaced equally in time, but encompass doses at non-equal intervals.

A dosing schedule of, for example, once/week, twice/week, threetimes/week, four times/week, five times/week, six times/week, seventimes/week, once every two weeks, once every three weeks, once everyfour weeks, once every five weeks, and the like, is available for theinvention. The dosing schedules encompass dosing for a total period oftime of, for example, one week, two weeks, three weeks, four weeks, fiveweeks, six weeks, two months, three months, four months, five months,six months, seven months, eight months, nine months, ten months, elevenmonths, and twelve months.

Provided are cycles of the above dosing schedules. The cycle can berepeated about, e.g., every seven days; every 14 days; every 21 days;every 28 days; every 35 days; 42 days; every 49 days; every 56 days;every 63 days; every 70 days; and the like. An interval of non-dosingcan occur between a cycle, where the interval can be about, e.g., sevendays; 14 days; 21 days; 28 days; 35 days; 42 days; 49 days; 56 days; 63days; 70 days; and the like. In this context, the term “about” meansplus or minus one day, plus or minus two days, plus or minus three days,plus or minus four days, plus or minus five days, plus or minus sixdays, or plus or minus seven days.

Cells derived from the methods of the present invention can becryopreserved using standard techniques in the art and stored for lateruse. Collections of cells derived from the methods of the presentinvention can be stored together in a cryopreserved cell and tissuebank.

Cells derived from the methods of the present invention may beformulated for administration according to any of the methods disclosedherein in any conventional manner using one or more physiologicallyacceptable carriers optionally comprising excipients and auxiliaries.Proper formulation is dependent upon the route of administration chosen.The compositions may also be administered to the individual in one ormore physiologically acceptable carriers. Carriers for cells mayinclude, but are not limited to, solutions of normal saline, phosphatebuffered saline (PBS), lactated Ringer's solution containing a mixtureof salts in physiologic concentrations, or cell culture medium.

The HSC populations of the invention and therapeutic agents comprisingthe same can be used to augment or replace bone marrow cells in bonemarrow transplantation. Human autologous and allogenic bone marrowtransplantation are currently used as therapies for diseases such asleukemia, lymphoma and other life-threatening disorders. The drawback ofthese procedures, however, is that a large amount of donor bone marrowmust be removed to ensure that there are enough cells for engraftment.

The HSC populations of the invention and therapeutic agents comprisingthe same can provide stem cells and progenitor cells that would reducethe need for large bone marrow donation. It would also be possible,according to the methods of the invention, to obtain a small marrowdonation and then expand the number of stem cells and progenitor cellsculturing and expanding in the placenta before infusion ortransplantation into a recipient. Alternatively, sufficient numbers ofHSCs can be obtained according to the methods of the present inventionusing only non-mobilized peripheral blood, thereby completelyeliminating the need for bone marrow donation altogether.

Compositions and methods of the present invention are useful in theexpansion of stem cells. In some embodiments, the expansion can be rapidcompared to traditional methods of expansion. In some embodiments,expansion may occur in the course of hours, days, or weeks (e.g.,selective expansion can occur for about 2 hours, 4 hours, 6 hours, 8hours, 12 hours, 16 hours, 20 hours, one day, two days, three days, fourdays, five days, six days, seven days, nine days, ten days, 11 days, 12days, 13 days, two weeks, three weeks, four weeks, or more. In someembodiments, a stem cell population may be expanded in terms of totalcell count by two-fold, three-fold, four-fold, five-fold, 6-fold,7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold,50-fold, 100-fold, 200-fold, 250-fold, 500-fold, 750-fold, 1000-fold,1250-fold, 1500-fold, 1750-fold, 2000-fold, or more. In someembodiments, a stem cell population may be expanded in terms of therelative number of cells with a stem cell phenotype in a broader cellpopulation (e.g. cells with a stem cell phenotype may make up about 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, 97.5%, 98%, 99%, or 100% of a cell population). Expansionmay be measured by a number of metrics including by doubling time forexample, by the amount of time it takes for a total cell number todouble (e.g., from 500 cells to 1,000 cells), or the time it takes for arelative percentage of the population to double (e.g., from 10% stemcells to 20% stem cells).

In another embodiment, the HSC populations of the invention andtherapeutic agents comprising the same can be used in a supplementaltreatment in addition to chemotherapy. Most chemotherapy agents used totarget and destroy cancer cells act by killing all proliferating cells,i.e., cells going through cell division. Since bone marrow is one of themost actively proliferating tissues in the body, hematopoietic stemcells are frequently damaged or destroyed by chemotherapy agents and inconsequence, blood cell production diminishes or ceases. Chemotherapymust be terminated at intervals to allow the patient's hematopoieticsystem to replenish the blood cell supply before resuming chemotherapy.It may take a month or more for the formerly quiescent stem cells toproliferate and increase the white blood cell count to acceptable levelsso that chemotherapy may resume (when again, the bone marrow stem cellsare destroyed).

While the blood cells regenerate between chemotherapy treatments,however, the cancer has time to grow and possibly become more resistantto the chemotherapy drugs due to natural selection. Therefore, thelonger chemotherapy is given and the shorter the duration betweentreatments, the greater the odds of successfully killing the cancer. Toshorten the time between chemotherapy treatments, the HSC populations ofthe invention and therapeutic agents comprising the same culturedaccording to the methods of the invention could be introduced into theindividual. Such treatment would reduce the time the individual wouldexhibit a low blood cell count, and would therefore permit earlierresumption of the chemotherapy treatment.

C. Methods for Producing a Cell Culture Medium

Further provided herein are methods for producing a cell culture medium(such as any of the cell culture media disclosed herein) for culturinghematopoietic stem cells (HSC). The method involves combining a base ora feed medium; and a compound of Formula I or a subembodiment disclosedherein. In some embodiments, the methods provided herein also includes aretinoic acid receptor (RAR) inhibitor or modulator. In someembodiments, the RAR inhibitor is ER50891. In additional embodiments,the method also includes combining one, two, three, or all four of stemcell factor (SCF), thrombopoietin (TPO), fms-related tyrosine kinase 3ligand (Flt31), and/or interleukin 6 (IL-6). The method can also includecombining one or more of a caspase inhibitor, a DNA methylationinhibitor, a p38 MAPK inhibitor, a GSK3 inhibitor, an RAR receptorantagonist, an inhibitor of the JAK/STAT pathway, and/or FBS (such as,heat inactivated FBS). In some embodiments, the methods provided hereindo not include a tetraspanin.

A “base medium,” as used herein, is a medium used for culturing cellswhich is, itself, directly used to culture the cells and is not used asan additive to other media, although various components may be added toa base medium. Examples of base media include, without limitation, DMEMmedium, IMDM medium, StemSpan Serum-Free Expansion Medium (SFEM),199/109 medium, Ham's F10/F12 medium, McCoy's 5A medium, Alpha MEMmedium (without and with phenol red), and RPMI 1640 medium. A basemedium may be modified, for example by the addition of salts, glucose,or other additives.

A “feed medium” is a medium used as a feed in a culture of a source ofCD34+ cells (e.g. bone marrow, cord blood, mobilized peripheral blood,and non-mobilized peripheral blood cells). A feed medium, like a basemedium, is designed with regard to the needs of the particular cellsbeing cultured. Thus, a base medium can be used as a basis for designinga feed medium. A feed medium can have higher concentrations of most, butnot all, components of a base culture medium. For example, somecomponents, such as salts, maybe kept at about 1× of the base mediumconcentration, as one would want to keep the feed isotonic with the basemedium. Thus, in some embodiments, various components are added to keepthe feed medium physiologic and others are added because they replenishnutrients to the cell culture. Other components, for example, nutrients,may be at about 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 12×, 14×, 16×, 20×,30×, 50×, 100× or more of their normal concentrations in a base medium.

V. Systems and Kits

Also provided herein are systems for maintaining and/or enhancing theexpansion of hematopoietic stem cells in culture. This system includes asource of CD34+ cells in culture (such as a CD34+ cells from one or moreof bone marrow, cord blood, mobilized peripheral blood, andnon-mobilized peripheral blood) and any of the cell culture mediacompositions described herein. In a particular embodiment, the system ofthe present invention maintains low oxygen culturing conditions. Assuch, the system provides an atmosphere to which the cultured cells areexposed having less than about 10% oxygen, such as any of about 10%,9.5, 9%, 8.5%, 8%, 7.5%, 7%, 6.5%, 6%, 5.5%, or 5%, 4.5%, 4%, 3.5%, 3%,2.75%, 2.5%, 2.25%, 2%, 1.75%, 1.5%%, 1.25%, 1%, 0.9%, 0.8%, 0.7%, 0.6%,or 0.5% or less oxygen. In some embodiments, the system provides anatmosphere to which the culture cells are exposed having any range inbetween 0.5% and 10% oxygen. Control of atmospheric oxygen in the systemcan be accomplished by any means known in the art, such as by additionof nitrogen.

In additional aspects, the invention disclosed herein provides one ormore kits. These kits can include either a base medium or a feed medium(such as, but not limited to, DMEM medium, IMDM medium, StemSpanSerum-Free Expansion Medium (SFEM), 199/109 medium, Ham's F10/F12medium, McCoy's 5A medium, Alpha MEM medium (without and with phenolred), and RPMI 1640 medium) as well as a compound of Formula I or asubembodiment disclosed herein. In some embodiments, the kits providedherein do not include a tetraspanin.

The kit can also include written instructions for maintaining and/orenhancing the expansion of HSCs in culture by culturing the cells usingthe kit's cell culture media components. The kit can also includeadditional components for inclusion into the cell culture media, such asone or more of thrombopoietin (TPO), stem cell factor (SCF),insulin-like growth factor 1 (IGF-1), erythroid differentiation factor(EDF), hepatocyte growth factor (HGF), epidermal growth factor (EGF),heat shock factor (HSF), pleiotrophin (PTN), basic fibroblast growthfactor (bFGF), angiopoietin 1 (ANG1), VEGF165, IL-10, laminin, caspaseinhibitor(s), epigallocatechin gallate (EGCG), Oct4-activating compound1 (OAC1), p38 MAPK inhibitor, JAK/STAT inhibitors, IL-3, IL-6, humangrowth hormone (HGH), fms-related tyrosine kinase 3 ligand (FLT3L),VEGF-C and ALK5/SMAD modulators or inhibitors, and fetal bovine serum(FBS) (including heat-inactivated FBS).

In some embodiments, the kit also includes a retinoic acid receptor(RAR) inhibitor or modulator. In some embodiments, the RAR inhibitor ormodulator is ER50891. In some embodiments, the kit includes alsothrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor1 (IGF-1), human growth hormone (HGH), fms-related tyrosine kinase 3ligand (FLT3L), and fetal bovine serum (FBS).

It is intended that every maximum numerical limitation given throughoutthis specification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The invention can be further understood by reference to the followingexamples, which are provided by way of illustration and are not meant tobe limiting.

VI. Particular Embodiments of the Present Disclosure Embodiment 1

A compound of Formula I

or a pharmaceutically acceptable salt, hydrate, or solvate thereof;wherein,

-   A is a fused cyclic moiety selected from the group consisting of a    phenyl, C₃₋₆ cycloalkyl, heterocycloalkyl, and heteroaryl;-   wherein each heterocycloalkyl comprises from 3 to 6 ring members    having 1 to 3 nitrogen atom ring members, and-   each heteroaryl comprises 5 to 6 ring members having 1 to 3 nitrogen    atom ring members;-   R¹ is selected from the group consisting of —C(O)—NR^(b)—R^(1a),    —NR^(b)—C(O)—R^(1a), —NR^(b)—C(O)—R^(1b), —NR^(b)—X¹—C(O)—R^(1a),    —C(O)—X¹—NR^(b)—R^(1a), —X¹—C(O)—NR^(b)—R^(1a),    —X¹—NR^(b)—C(O)—R^(1a), —NR^(b)—C(O)—X¹—C(O)—R^(1b),    —C(O)—NR^(b)—X¹—C(O)—R^(b), —NR^(b)—C(O)—O—R^(1a),    —O—C(O)—NR^(b)—R^(1a), —X¹—NR^(b)—C(O)—O—R^(1a),    —X¹—O—C(O)—NR^(b)—R^(1a), —NR^(b)—R^(1a), and —C(O)—R^(1a);-   R^(1a) is selected from the group consisting of H, C₁₋₁₀ alkyl;    C₁₋₁₀ haloalkyl;-   R^(1b) is selected from the group consisting of —OR^(a),    —NR^(a)R^(b),-   each R² is independently selected from the group consisting of    halogen, —CN, —C₁₋₈ alkyl, —C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₁₋₈    haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈ alkoxy, —C(O)—R^(2a),    —NR^(b)—C(O)—R^(2a), —SR^(a), —X¹—SR^(a), —OR^(a), —X¹—OR^(a),    —NR^(a)R^(b), —X¹—NR^(a)R^(b), —S(O)₂R^(a), —S(O)₂NR^(a)R^(b),    —X¹—S(O)₂R^(a), and —X¹—S(O)₂NR^(a)R^(b)-   each R³ is independently selected from the group consisting of    halogen, —CN, —C₁₋₈ alkyl, —C₂₋₈ alkenyl, C₂₋₈ alkynyl, C₁₋₈    haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈ alkoxy, —C(O)—R^(3a), —SR^(a),    —X¹—SR^(a), —OR^(a), —X¹—OR^(a), —NR^(a)R^(b), —X¹—NR^(a)R^(b),    —S(O)₂R^(a), —S(O)₂NR^(a)R^(b), —X¹—S(O)₂R^(a), and    —X¹—S(O)₂NR^(a)R^(b);-   each R^(2a) and R^(3a) is independently selected from the group    consisting of H, C₁₋₁₀ alkyl, C₁₋₁₀ haloalkyl, —OR^(a), —X¹—OR^(a),    —NR^(a)R^(b), and —X¹—NR^(a)R^(b);-   R^(4a) is selected from the group consisting of —OR^(a), and    —NR^(a)R^(b);-   R^(4b) is H; or R^(4a) and R^(4b) are combined to form an oxo or an    oxime moiety;-   each R^(a) and R^(b) is independently selected from the group    consisting of H and C₁₋₄ alkyl;-   each X¹ is C₁₋₄ alkylene;-   the subscript n is an integer from 0 to 3; and-   the subscript m is an integer from 0 to 2.

Embodiment 2

The compound of embodiment 1, wherein

-   A is a fused cyclic moiety selected from the group consisting of a    C₃₋₆ cycloalkyl, heterocycloalkyl, and phenyl,-   wherein each heterocycloalkyl comprises from 3 to 6 ring members    having 1 to 3 nitrogen atom ring members.

Embodiment 3

The compound of embodiment 1, wherein

-   A is a fused cyclic moiety selected from the group consisting of a    C₃₋₆ cycloalkyl and phenyl.

Embodiment 4

The compound of embodiment 1, wherein

-   A is a fused C₃₋₆ cycloalkyl.

Embodiment 5

The compound of any one of embodiments 1 to 4, wherein

-   R^(4a) is —OR^(a); R^(4b) is H; or R^(4a) and R^(4b) are combined to    form an oxo moiety

Embodiment 6

The compound of any one of embodiments 1 to 4, wherein

-   R^(4a) is —OR^(a); and R^(4b) is H.

Embodiment 7

The compound of any one of embodiments 1 to 4, wherein

-   R^(4a) is —NR^(a)R^(b); and R^(4b) is H.

Embodiment 8

The compound of any one of embodiments 1 to 7, wherein

-   R¹ is selected from the group consisting of —C(O)—NR^(b)—R^(1a),    —NR^(b)—C(O)—R^(a), —NR^(b)-X¹_C(O)—R^(a), —C(O)—X¹—NR^(b)—R^(a),    —X¹—C(O)—NR^(b)—R^(a), —X¹—NR^(b)—C(O)—R^(1a),    —NR^(b)—C(O)—X¹—C(O)—R^(1b), —C(O)—NR^(b)—X¹—C(O)—R^(1b),    —NR^(b)—C(O)—O—R^(1a), —O—C(O)—NR^(b)—R^(1a), —NR^(b)—R^(1a), and    —C(O)—R^(1a).

Embodiment 9

The compound any one of embodiments 1 to 7, wherein

-   R¹ is selected from the group consisting of —C(O)—NH—R^(a),    —NH—C(O)—R^(a), —NH—C(O)—O—R^(a), —O—C(O)—NH—R^(a), —NH—R^(1a), and    —C(O)—R^(1a).

Embodiment 10

The compound any one of embodiments 1 to 7, wherein

-   R¹ is selected from the group consisting of —NH—C(O)—R^(a),    —NH—C(O)—O—R^(a), and —NR^(b)—R^(1a)

Embodiment 11

The compound any one of embodiments 1 to 7, wherein

-   R¹ is selected from the group consisting of —NH—C(O)—R^(1a), and    —NH—C(O)—O—R^(a)

Embodiment 12

The compound of any one of embodiments 1 to 7, wherein

-   R¹ is —NH—C(O)—R^(1a).

Embodiment 13

The compound of any one of embodiments 1 to 12, wherein

-   each R² is independently selected from the group consisting of    halogen, —C₁₋₈ alkyl, C₁₋₈ haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈ alkoxy,    —C(O)—R^(2a), —NR^(b)—C(O)—R^(2a)—SR^(a), —X¹—SR^(a), —OR^(a),    —X¹—OR^(a), —NR^(a)R^(b), and —X¹—NR^(a)R^(b)

Embodiment 14

The compound of any one of embodiments 1 to 13, wherein

-   each R³ is independently selected from the group consisting of    halogen, —C₁₋₈ alkyl, —C₁₋₈ haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈    alkoxy, —C(O)—R^(3a), —SR^(a), —X¹—SR^(a), —OR^(a), —X¹—OR^(a),    —NR^(a)R^(b), —X¹—NR^(a)R^(b), —S(O)₂R^(a), —S(O)₂NR^(a)R^(b),    —X¹—S(O)₂R^(a), and —X¹—S(O)₂NR^(a)R^(b).

Embodiment 15

The compound of any one of embodiments 1 to 14, wherein

-   each R² and R³ is independently selected from the group consisting    of halogen, —C₁₋₈ alkyl, C₁₋₈ haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈    alkoxy, —OR^(a), —X¹—OR^(a), —NR^(a)R^(b), —X¹—NR^(a)R^(b),    —S(O)₂R^(a), —S(O)₂NR^(a)R^(b), —X¹—S(O)₂R^(a), and    —X¹—S(O)₂NR^(a)R^(b).

Embodiment 16

The compound of any one of embodiments 1 to 14, wherein

-   each R² and R³ is independently selected from the group consisting    of halogen, —C₁₋₈ alkyl, C₁₋₈ haloalkyl, —C₁₋₈ alkoxy, —X¹—C₁₋₈    alkoxy, —OR^(a), —NR^(b)—C(O)—R^(2a)—X¹—OR^(a), —NR^(a)R^(b), and    —X¹—NR^(a)R^(b).

Embodiment 17

The compound of any one of embodiments 1 to 14, wherein

-   each R² and R³ is independently selected from the group consisting    of halogen, —C₁₋₈ alkyl, C₁₋₈ haloalkyl, —OR^(a), —X¹—OR^(a),    —NR^(a)R^(b), and —X¹—NR^(a)R^(b).

Embodiment 18

The compound of any one of embodiments 1 to 14, wherein

-   each R² and R³ is independently selected from the group consisting    of —OR^(a), —X¹—OR^(a), —NR^(a)R^(b) or —X¹—NR^(a)R^(b).

Embodiment 19

The compound of any one of embodiments 1 to 18, wherein R^(1a) is C₁₋₆alkyl or C₁₋₆ haloalkyl.

Embodiment 20

The compound of any one of embodiments 1 to 18, wherein R^(1a) is C₁₋₆alkyl.

Embodiment 21

The compound of any one of embodiments 1 to 18, wherein R^(1b) is—OR^(a).

Embodiment 22

The compound of any one of embodiments 1 to 18, wherein R^(1b) is —OH.

Embodiment 23

The compound of any one of embodiments 1 to 22, wherein each R^(a) andR^(b) is independently selected from the group consisting of H and C₁₋₂alkyl.

Embodiment 24

The compound of any one of embodiments 1 to 23, wherein each X¹ is C₁₋₂alkylene.

Embodiment 25

The compound of any one of embodiments 1 to 23, wherein each X¹ is C₁alkylene.

Embodiment 26

The compound of any one of embodiments 1 to 25, wherein the subscript nis an integer from 1 to 3.

Embodiment 27

The compound of any one of embodiments 1 to 25, wherein the subscript nis 1.

Embodiment 28

The compound of any one of embodiments 1 to 25, wherein the subscript nis 0.

Embodiment 29

The compound of any one of embodiments 1 to 28, wherein the subscript mis an integer from 1 to 2.

Embodiment 30

The compound of any one of embodiments 1 to 28, wherein the subscript mis 0.

Embodiment 31

The compound of any one of embodiments 1 to 28, wherein the subscript mis 1.

Embodiment 32

The compound of any one of embodiments 1 to 31, wherein the compound ofFormula I has the structure of Formula I-1 or I-2

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

Embodiment 33

The compound of any one of embodiments 1, or 8 to 31, wherein thecompound of Formula I has the structure of Formula Ia

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 34

The compound of embodiment 33, wherein the compound of Formula Ia hasthe structure of Formula Ia′

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 35

The compound of embodiment 33, wherein the compound of Formula Ia hasthe structure of Formula Ia1 or Ia2

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

Embodiment 36

The compound of embodiment 35, wherein the compound of Formula Ia1 orIa2 has the structure of Formula Ia1′ or Ia2′

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 37

The compound of any one of embodiments 1, or 8 to 31, wherein thecompound of Formula I has the structure of Formula Ib or Ic

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 38

The compound of embodiment 37, wherein the compounds of Formula Ib hasthe structure of Formula Ib1 or Ib2.

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

Embodiment 39

The compound of embodiment 37, wherein the compounds of Formula Ic hasthe structure of Formula Ic1 or Ic2.

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,whereinR^(4a) is selected from the group consisting of —OR^(a), and—NR^(a)R^(b);R^(4b) is H.

Embodiment 40

The compound of any one of embodiments 32 to 39, wherein R^(4a) is —OHor —NH₂.

Embodiment 41

The compound of any one of embodiments 32 to 39, wherein R^(4a) is —OH.

Embodiment 42

A compound of any one of embodiments 1 to 31, where the compound ofFormula I has the structure of Formula II

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 43

The compound of embodiment 42, wherein

-   R¹ is selected from the group consisting of —NH—C(O)—R^(1a),    —NH—C(O)—O—R^(1a); —NH—X¹—C(O)—R^(1a), and —NH—R^(1a);-   each R² and R³ is independently selected from the group consisting    of —NH₂, —OH, —X¹—NH₂, —X¹—OH;-   R¹ is selected from the group consisting of C₁₋₆ alkyl; and C₁₋₆    haloalkyl;-   each X¹ is C₁₋₂ alkylene;-   the subscript n is an integer from 0 to 2; and-   the subscript m is 0 or 1.

Embodiment 44

The compound of embodiment 42 or embodiment 43, wherein the compound ofFormula II has the structure of Formula IIa

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 45

The compound of embodiment 44, wherein the compound of Formula IIa hasthe structure of Formula IIa′

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 46

The compound of embodiment 44, wherein the compound of Formula IIa hasthe structure of Formula IIa1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 47

The compound of embodiment 45 or embodiment 46, wherein

R¹ is selected from the group consisting of —NH—C(O)—R^(1a);

R² is independently selected from the group consisting of —NH₂ or —OH;

R^(1a) is selected from the group consisting of C₁₋₆ alkyl; and C₁₋₆haloalkyl; and

the subscript n is 0 or 1.

Embodiment 48

The compound of embodiment 42 or embodiment 43, wherein the compound ofFormula II has the structure of Formula IIb or IIc

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 49

The compound of embodiment 48, wherein the compound of Formula IIb orIIc has the structure of Formula IIb1 or IIc1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof.

Embodiment 50

The compound of embodiment 49, wherein

R¹ is selected from the group consisting of —NH—C(O)—R^(1a);

R² is independently selected from the group consisting of —NH₂ or —OH;

R^(1a) is selected from the group consisting of C₁₋₆ alkyl; and C₁₋₆haloalkyl; and

the subscript n is 0 or 1.

Embodiment 51

The compound of embodiment 1, wherein said compound is selected fromTable 1.

Embodiment 52

A method for expanding hematopoietic stem cells in culture, the methodcomprising contacting a source of CD34+ cells in culture with aneffective amount of a compound of any one of embodiments 1-51, therebyexpanding hematopoietic stem cells in the culture.

Embodiment 53

The method of embodiment 52, wherein the source of CD34+ cells isselected from the group consisting of bone marrow, cord blood, mobilizedperipheral blood, and non-mobilized peripheral blood.

Embodiment 54

The method of embodiment 52, wherein the source of CD34+ cells isnon-mobilized peripheral blood.

Embodiment 55

The method of embodiment 53 or embodiment 54, wherein the source ofCD34+ cells comprises one or more of (a) CD34+ hematopoieticprogenitors; (b) CD34+ early hematopoietic progenitors and/or stemcells; (c) CD133+ early hematopoietic progenitors and/or stem cells;and/or (d) CD90+ early hematopoietic progenitors and/or stem cells.

Embodiment 56

The method of any one of embodiments 52-55, further comprising aretinoic acid receptor (RAR) inhibitor or modulator.

Embodiment 57

The method of embodiment 11, wherein the retinoic acid receptor (RAR)inhibitor or modulator is ER50891.

Embodiment 58

The method of any one of embodiments 52-57, wherein the method furthercomprises culturing the cells under low oxygen conditions.

Embodiment 59

The method of embodiment 58, wherein low oxygen conditions comprise anatmosphere containing about 5% oxygen or less.

Embodiment 60

The method of any one of embodiments 52-59, wherein the method furthercomprises contacting the cells with one or more agents selected from thegroup consisting of thrombopoietin (TPO), stem cell factor (SCF),hepatocyte growth factor (HGF), p38 MAPK inhibitor, epidermal growthfactor (EGF), JAK/STAT inhibitors, IL-3, IL-6 human growth hormone(HGH), fms-related tyrosine kinase 3 ligand (FLT3L), VEGF-C andALK5/SMAD modulators or inhibitors.

Embodiment 61

The method of any one of embodiments 52-59, wherein the method furthercomprises contacting the cells with thrombopoietin (TPO), stem cellfactor (SCF), and fms-related tyrosine kinase 3 ligand (FLT3L).

Embodiment 62

The method of any one of embodiments 52-59, wherein the method furthercomprises contacting the cells with thrombopoietin (TPO) and stem cellfactor (SCF).

Embodiment 63

The method of any one of embodiments 52-62, wherein said methodstabilizes the hematopoietic stem cell phenotype.

Embodiment 64

The method of embodiments 63, wherein the hematopoietic stem cellphenotype comprises CD45+, CD34+, CD133+, CD90+, CD45RA−, CD38 low/−,and negative for major hematopoietic lineage markers including CD2, CD3,CD4, CD5, CD8, CD14, CD16, CD19, CD20, CD56.

Embodiment 65

The method of any one of embodiments 52-64, wherein CD133+ and/or CD90+positive cells are increased compared to cells in culture that are notcontacted with a compound of any one of embodiments 1-51.

Embodiment 66

The method of embodiment 65, wherein the cells exhibit at least abouttwo times the number of CD133+ and/or CD90+ positive cells compared tocells in culture that are not contacted with a compound of any one ofembodiments 1-51 after 7 day in culture.

Embodiment 67

The method of any one of embodiments 52-66, wherein the source of theCD34+ cells is a human being.

Embodiment 68

A medium for expanding hematopoietic stem cells in culture comprising:

-   -   (a) (i) a base medium or (ii) a feed medium; and    -   (b) a compound of any one of embodiments 1-51.

Embodiment 69

The medium of embodiment 68, wherein the medium further comprises (c) aretinoic acid receptor (RAR) inhibitor or modulator.

Embodiment 70

The medium of embodiment 69, wherein the retinoic acid receptor (RAR)inhibitor or modulator is ER50891.

Embodiment 71

The medium of any one of embodiments 68-70, wherein the medium furthercomprises (c) one or more agents selected from the group consisting ofthrombopoietin (TPO), stem cell factor (SCF), insulin-like growth factor1 (IGF-1), erythroid differentiation factor (EDF), hepatocyte growthfactor (HGF), epidermal growth factor (EGF), heat shock factor (HSF),pleiotrophin (PTN), basic fibroblast growth factor (bFGF), angiopoietin1 (ANG1), VEGF165, IL-10, laminin, caspase inhibitor(s),epigallocatechin gallate (EGCG), Oct4-activating compound 1 (OAC1), p38MAPK inhibitor, JAK/STAT inhibitors, IL-3, IL-6, human growth hormone(HGH), fms-related tyrosine kinase 3 ligand (FLT3L), VEGF-C andALK5/SMAD modulators or inhibitors, and fetal bovine serum (FBS).

Embodiment 72

The medium of embodiment 71, wherein the FBS is heat inactivated.

Embodiment 73

The medium of any one of embodiments 68-70, wherein the medium furthercomprises (c) thrombopoietin (TPO), stem cell factor (SCF), andfms-related tyrosine kinase 3 ligand (FLT3L).

Embodiment 74

The medium of any one of embodiments 68-70, wherein the medium furthercomprises (c) thrombopoietin (TPO) and stem cell factor (SCF).

Embodiment 75

The medium of any one of embodiments 68-74, wherein the base medium is abase salt medium.

Embodiment 76

The medium of embodiment 72, wherein the base salt medium is alpha MEM.

Embodiment 77

The medium of embodiment 75, wherein the base salt medium comprises asufficient amount of CaCl₂ to adjust the base salt medium to 320-380mOsm.

Embodiment 78

A method for expanding hematopoietic stem cells in culture, the methodcomprising contacting a source of CD34+ cells in culture with the mediumof any one of embodiments 68-77, thereby expanding hematopoietic stemcells in the culture.

Embodiment 79

A system for expanding hematopoietic stem cells in culture, the systemcomprising (a) a source of CD34+ cells in culture; and (b) the medium ofany one of embodiments 68-77.

Embodiment 80

The system of embodiment 79, wherein the source of CD34+ cells isselected from the group consisting of bone marrow, cord blood, mobilizedperipheral blood, and non-mobilized peripheral blood.

Embodiment 81

The system of embodiment 80, wherein the source of CD34+ cells isnon-mobilized peripheral blood.

Embodiment 82

The system of embodiment 80 or embodiment 81, wherein the source ofCD34+ cells comprises one or more of (a) CD34+ hematopoieticprogenitors; (b) CD34+ early hematopoietic progenitors and/or stemcells; (c) CD133+ early hematopoietic progenitors and/or stem cells;and/or (d) CD90+ early hematopoietic progenitors and/or stem cells.

Embodiment 83

The system of any one of embodiments 79-82, further comprising (c) anatmosphere containing low oxygen.

Embodiment 84

The system of embodiment 83, wherein the atmosphere contains about 5%oxygen or less.

Embodiment 85

The system of any one of embodiments 79-84, wherein the source of CD34+cells is a human being.

Embodiment 86

A kit comprising:

(a) (i) a base medium or (ii) a feed medium; and

(b) a compound of any one of embodiments 1-51.

Embodiment 87

The kit of embodiment 86, further comprising (c) written instructionsfor maintaining and/or expanding hematopoietic stem cells in culture.

Embodiment 88

The kit of embodiment 86 or embodiment 87, further comprising (d) aretinoic acid receptor (RAR) inhibitor or modulator.

Embodiment 89

The medium of embodiment 88, wherein the retinoic acid receptor (RAR)inhibitor or modulator is ER50891.

Embodiment 90

The kit of embodiment 86-89, further comprising one or more agentsselected from the group consisting of thrombopoietin (TPO), stem cellfactor (SCF), insulin-like growth factor 1 (IGF-1), erythroiddifferentiation factor (EDF), hepatocyte growth factor (HGF), epidermalgrowth factor (EGF), heat shock factor (HSF), pleiotrophin (PTN), basicfibroblast growth factor (bFGF), angiopoietin 1 (ANG1), VEGF165, IL-10,laminin, caspase inhibitor(s), epigallocatechin gallate (EGCG),Oct4-activating compound 1 (OAC1), p38 MAPK inhibitor, JAK/STATinhibitors, IL-3, IL-6 human growth hormone (HGH), fms-related tyrosinekinase 3 ligand (FLT3L), VEGF-C and ALK5/SMAD modulators or inhibitors,and fetal bovine serum (FBS).

Embodiment 91

The kit of embodiment 90, wherein the FBS is heat inactivated.

Embodiment 92

The kit of any one of embodiments 86-89, further comprising (d)thrombopoietin (TPO), stem cell factor (SCF), and fms-related tyrosinekinase 3 ligand (FLT3L).

Embodiment 93

The kit of any one of embodiments 86-89, further comprising (d)thrombopoietin (TPO) and stem cell factor (SCF).

Embodiment 94

The kit of any one of embodiments 86-93, wherein the base medium is abase salt medium.

Embodiment 95

The kit of embodiment 94, wherein the base salt medium is alpha MEM.

Embodiment 96

The kit of embodiment 94, wherein the base salt medium comprises320-380mOsm CaCl₂.

Embodiment 97

A population of hematopoietic stem cells produced by the method of anyone of embodiment 52-67 or 78.

Embodiment 98

A therapeutic agent comprising the population of hematopoietic stemcells of embodiment 97.

Embodiment 99

A method of treating an individual in need of hematopoieticreconstitution, comprising administering to said individual thetherapeutic agent of embodiment 98.

Embodiment 100

The method of embodiment 99, wherein the individual is a bone marrowdonor or recipient.

Embodiment 101

The method of embodiment 100, wherein the individual is diagnosed withcancer.

Embodiment 102

The method of embodiment 101, wherein the method is used as asupplemental treatment in addition to chemotherapy.

Embodiment 103

The method of embodiment 102, wherein the method is used to shorten thetime between chemotherapy treatments.

Embodiment 104

The method of embodiment 99, wherein the individual is diagnosed with anautoimmune disease.

Embodiment 105

A method for producing a cell culture media for culturing hematopoieticstem cells (HSC), the method comprising: combining (a) a base or a feedmedium; and (b) a compound of any one of embodiment 1-51.

Embodiment 106

The method of embodiment 105, further comprising (c) a retinoic acidreceptor (RAR) inhibitor or modulator.

Embodiment 107

The method of embodiment 106, wherein the retinoic acid receptor (RAR)inhibitor or modulator is ER50891.

Embodiment 108

The method of any one of embodiment 105-107, further comprisingthrombopoietin (TPO), stem cell factor (SCF), and/or fms-relatedtyrosine kinase 3 ligand (FLT3L).

Embodiment 109

The method of any one of embodiment 105-107, further comprisingthrombopoietin (TPO) and stem cell factor (SCF).

Embodiment 110

The method of any one of embodiments 105-109, further comprisingcombining one or more of insulin-like growth factor 1 (IGF-1), erythroiddifferentiation factor (EDF), hepatocyte growth factor (HGF), epidermalgrowth factor (EGF), heat shock factor (HSF), pleiotrophin (PTN), basicfibroblast growth factor (bFGF), angiopoietin 1 (ANG1), VEGF165, IL-10,laminin, caspase inhibitor(s), epigallocatechin gallate (EGCG),Oct4-activating compound 1 (OAC1), p38 MAPK inhibitor, JAK/STATinhibitors, IL-3, IL-6 human growth hormone (HGH), fms-related tyrosinekinase 3 ligand (FLT3L), VEGF-C and ALK5/SMAD modulators or inhibitors,and fetal bovine serum (FBS).

Embodiment 111

The method of embodiment 110, wherein the FBS is heat-inactivated FBS.

Embodiment 112

The method of any one of embodiments 105-108, further comprising (d)insulin-like growth factor 1 (IGF-1), human growth hormone (HGH), andfetal bovine serum (FBS).

Embodiment 113

The method of any one of embodiments 105-112, wherein the base or feedmedium is Alpha MEM.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Reagents and solvents used below can be obtained from commercial sourcessuch as MilliporeSigma (St. Louis, Mo., USA).

¹H-NMR spectra were recorded on a Varian Mercury 400 MHz NMRspectrometer. Chemical shifts were internally referenced to the residualproton resonance in CDCl3 (7.26 ppm) and are tabulated in the order:multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet) and number of protons. ¹³C NMR was recorded at 100 MHz.Proton. Carbon chemical shifts were internally referenced to thedeuterated solvent signals in CDCl3 (77.20 ppm).

Mass spectrometry results are reported as the ratio of mass over charge,followed by the relative abundance of each ion (in parenthesis). In theexamples, a single m/z value is reported for the M+H (or, as noted, M−H)ion containing the most common atomic isotopes. Isotope patternscorrespond to the expected formula in all cases. Electrospray ionization(ESI) mass spectrometry analysis was conducted on a Shimadzu LC-MS2020using Agilent C18 column (Eclipse XDB-C18, 5 um, 2.1×50 mm) with flowrate of 1 mL/min. Mobile phase A: 0.1% of formic acid in water; mobilephase B: 0.1% of formic acid in acetonitrile. Normally the analyte wasdissolved in methanol at 0.1 mg/mL and 1 microliter was infused with thedelivery solvent into the mass spectrometer, which scanned from 100 to1500 daltons. All compounds could be analyzed in the positive ESI mode,or analyzed in the negative ESI mode.

Analytical HPLC was performed on Agilent 1200 HPLC with a Zorbax EclipseXDB C18 column (2.1×150 mm) with flow rate of 1 mL/min. Mobile phase A:0.1% of TFA in water; mobile phase B: 0.1% of TFA in acetonitrile.

Preparative HPLC was performed on Varian ProStar using Hamilton C18PRP-1 column (15×250 mm) with flow rate of 20 mL/min. Mobile phase A:0.1% of TFA in water; mobile phase B: 0.1% of TFA in acetonitrile.

The following abbreviations are used in the Examples and throughout thedescription of the invention:

THF: Tetrahydrofuran

TLC: Thin layer chromatography

TFA: Trifluoroacetic Acid

TEA: Triethylamine

Tol: Toluene

DCM: Dichloromethane

DCE: 1,2-dichloroethane

DMF: Dimethyl formamide

DMSO: Dimethyl sulfoxide

DPPA: Diphenylphosphoryl azide

MeOH: Methanol

BINAP: (2,2′-bis(diphenylphosphino)-1,1′-binaphthyl)

Pd₂(dba)₃: Tris(dibenzylideneacetone)dipalladium(O)

PPA Polyphosphoric acid

PDC Pyridinium dichromate (Cornforth reagent)

PE: Petroleum ether

EA: Ethyl acetate

XPhos: 2-Dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

LCMS: Liquid Chromatography-Mass Spectrometry

HPLC: High Pressure Liquid Chromatography

t-Bu: tert-butyl

Et Ethyl

OAc: Acetate

Piv Pivalyl (t-BuC(O)—)

Compounds within the scope of this invention can be synthesized asdescribed below, using a variety of reactions known to the skilledartisan. One skilled in the art will also recognize that alternativemethods may be employed to synthesize the target compounds of thisinvention, and that the approaches described within the body of thisdocument are not exhaustive, but do provide broadly applicable andpractical routes to compounds of interest.

Certain molecules claimed in this patent can exist in differentenantiomeric and diastereomeric forms and all such variants of thesecompounds are within the scope of the present disclosure.

The detailed description of the experimental procedures used tosynthesize key compounds in this text lead to molecules that aredescribed by the physical data identifying them as well as by thestructural depictions associated with them.

Those skilled in the art will also recognize that during standard workup procedures in organic chemistry, acids and bases are frequently used.Salts of the parent compounds are sometimes produced, if they possessthe necessary intrinsic acidity or basicity, during the experimentalprocedures described within this patent.

Example 1: Synthesis ofN-(8-oxo-1,2,3,3a,8,8a-hexahydrocyclopenta[a]inden-6-yl)pivalamide(Compound 1.001)

A mixture of compound 1.1 (4.9 g, 437 mmol, 1.0 eq) in benzene (50 mL)and AlCl₃ (17.5 g, 1311 mmol, 3.0 eq) was added 3 times then heated atreflux for 3 h. The reaction was quenched by 3 M HCl and the aqueoussolution was extracted with ethyl acetate. The combined organic layerwas dried and concentrated to a residue which was purified by columnchromatography (PE/EA=100:1) to give compound 1.2 (3.4 g, 45%).

A mixture of compound 1.2 (3.4 g, 19.7 mmol, 1.0 eq) in conc. HNO₃ (32mL) and conc. H₂SO₄ (4 mL) was heated at 80° C. for 1 h. Water was addedand the crude mixture was extracted with ethyl acetate. The combinedorganic layer was dried and concentrated to a residue which was purifiedby column chromatography (PE/EA=30:1) to give compound 1.3 (2.7 g, 63%)as yellow solid.

To a mixture of 1.3 (2.7 g, 12.44 mmol, 1.0 eq), iron powder (3.5 g,62.2 mmol, 5.0 eq), NH₄Cl (6.65 g, 10.0 mmol, 10.0 eq) in ethanol/water(v/v=2:1, 20 mL/10 mL) was stirred at 80° C. for 1 h under nitrogenatmosphere. After the reaction completely, the solid was filtered outand the filtrate was concentrated in vacuo to provide 1.4 (1.8 g, 77%).

To a mixture of 1.4 (50 mg, 0.267 mmol, 1.0 eq) in THF (5 mL) was addedNa₂CO₃ (114 mg, 1.07 mmol, 4.0 eq) and 1.5 (65 mg, 0.535 mmol, 2.0 eq).The mixture was stirred at rt for 30 min under nitrogen atmosphere. Thenthe mixture was filtered, added H₂O (3 mL), extracted with EA (2×9 mL).The residue was dried over Na₂SO₄ and concentrated under reducedpressure to give a residue which was purified by Prep- to give Compound1.001 (40 mg, 56%) as white solid. LCMS: [M+1]=272. ¹H NMR (400 MHz,DMSO): δ 9.32 (s, 1H), 8.09 (s, 1H), 8.08-7.82 (m, 1H), 7.31-7.29 (m,1H), 3.39-3.37 (m, 1H), 3.01-2.98 (m, 1H), 2.51-2.50 (m, 2H), 2.19-2.10(m, 1H), 1.84-1.79 (m, 1H), 1.78-1.40 (m, 2H), 1.12 (s, 9H).

Example 2: Synthesis ofN-(9-oxo-2,3,4,4a,9,9a-hexahydro-1H-fluoren-7-yl)pivalamide (Compound1.002)

A mixture of compound 2.1 (400 mg, 3.2 mmol, 1.0 eq) and AlCl₃ (1.27 g,9.5 mmol, 3.0 eq) in benzene (10 mL) was heated at reflux for 2 h. Thereaction was quenched by 3 M HCl and the aqueous solution was extractedwith ethyl acetate. The combined organic layer was dried andconcentrated to a residue which was purified by column chromatography(PE/EA=100:1) to give compound 2.2 (150 mg, 25%).

A mixture of compound 2.2 (140 mg, 0.75 mmol, 1.0 eq) in conc. HNO₃ (1.3mL) and conc. H₂SO₄ (0.16 mL) was heated at 80° C. for 2 h. Water wasadded and the crude mixture was extracted with ethyl acetate. Thecombined organic layer was dried and concentrated to a residue which waspurified by column chromatography (PE/EA=30:1) to give compound 2.3 (51mg, 29%) as white solid.

To a mixture of 2.3 (51 mg, 0.22 mmol, 1.0 eq), iron powder (62 mg, 1.1mmol, 5.0 eq) NH₄Cl (118 mg, 2.2 mmol, 10.0 eq) in ethanol/water(v/v=2:1, 5 mL/2.5 mL) was stirred at 80° C. for 1 h under nitrogenatmosphere. After the reaction completely, the solid was filtered outand the filtrate was concentrated in vacuo to provide 2.4 (30 mg, 68%).

To a mixture of 2.4 (30 mg, 0.15 mmol, 1.0 eq) in THF (3 mL) was addedNa₂CO₃ (63.6 mg, 0.60 mmol, 4.0 eq) and 2.5 (36 mg, 0.30 mmol, 2.0 eq).The mixture was stirred at rt for 30 min under nitrogen atmosphere. Thenthe mixture was filtered, added H₂O (5 mL), extracted with EA (5×3 mL).The residue was dried over Na₂SO₄ and concentrated under reducedpressure to give a residue which was purified by Prep-TLC to giveCompound 1.002 (12 mg, 28%) as white solid. LCMS: [M+1]=286. ¹H NMR (400MHz, CDCl₃): δ 9.36 (s, 1H), 8.14 (m, 1H), 7.91-7.85 (m, 1H), 7.30-7.27(m, 1H), 3.15 (s, 1H), 2.61 (s, 1H), 2.18-2.21 (m, 1H), 1.74-1.72 (m,4H), 1.58-1.53 (m, 3H), 1.23 (s, 9H).

Example 3: Synthesis of tert-butyl (9-oxo-9H-fluoren-2-yl)carbamate(Compound 1.003)

To a mixture of compound 3.1 (224 mg, 1 mmol, 1.0 eq), Et₃N (158 mg,1.55 mmol, 1.55 eq) and t-BuOH (120 mg, 1.62 mmol, 1.62 eq) in toluene(100 mL) was added DPPA (413 mg, 1.5 mmol, 1.5 eq) at rt. The mixturewas refluxed at 105° C. for 1 h. The reaction was monitored by LCMS. Thereaction mixture was diluted with water (20 mL), filtered. The filtratewas extracted with EA (2×20 mL). The organic layers were combined washedwith water (30 mL), brine (30 mL), dried, filtered and concentrated togive a residue which purified by Prep-TLC (PE/EA=5:1) to give Compound1.003 (54 mg, 18%) as yellow solid. LCMS: [M+Na]=318. ¹H NMR (400 MHz,CDCl₃): δ 9.67 (s, 1H), 7.76 (s, 1H), 7.75-7.63 (m, 2H), 7.59-7.52 (m,3H), 7.29-7.25 (m, 1H), 1.47 (s, 9H).

Example 4: Synthesis of 2-(tert-butylamino)-9H-fluoren-9-one (Compound1.004)

To a mixture of compound 4.1 (200 mg, 0.772 mmol, 1.0 eq) in PhMe (5 mL)was added compound 12 (67 mg, 0.927 mmol, 1.2 eq), Pd₂(dba)₃ (1.3 mg,0.00579 mmol, 0.0075 eq), BINAP (1.2 mg, 0.00193 mmol, 0.0025 eq) andNaOtBu (104 mg, 1.08 mmol, 1.4 eq). The mixture was microwaved at 100°C. for 30 min. The reaction was monitored by LCMS. Then the mixture wasquenched with water (5 mL). The precipitated solid was filtered, washedwith THF (5 mL). The residue was purified by Prep-HPLC to give Compound1.004 (5 mg, 3%) as an orange solid. LCMS: [M+1]=252. ¹H NMR (400 MHz,DMSO-d₆): δ 7.50-7.35 (m, 4H), 7.15-7.10 (m, 1H), 6.92 (s, 1H), 6.83 (d,J=8.0 Hz, 1H), 5.83 (s, 1H), 1.32 (s, 9H).

Example 5: Synthesis of N-(9-oxo-9H-fluoren-2-yl)pivalamide (Compound1.005)

To a mixture of compound 5.1 (1.5 g, 7.7 mmol, 1.0 eq) and TEA (2.33 g,23 mmol, 3.0 eq) in DCM (50 mL) was added compound 5 (1.1 g, 9 mmol, 1.2eq) at 0° C. under nitrogen atmosphere. The mixture was stirred at rtfor 1 h. The reaction was monitored by TLC. Then the mixture wasfiltered, added H₂O (20 mL), extracted with DCM (3×50 mL). The residuewas treated with EA and filtered to give Compound 1.005 (1.7 g, 79%) asan orange solid. TLC: DCM:MeOH=20:1, UV 254 nm. Rf (compound 5.1)=0.3.Rf (Compound 1.005)=0.8. LCMS: [M+1]=280. ¹H NMR (400 MHz, DMSO-d₆): δ9.43 (s, 1H), 7.98 (s, 1H), 7.85-7.83 (m, 1H), 7.73-7.69 (m, 2H),7.60-7.55 (m, 2H), 7.35-7.26 (m, 1H), 1.24 (s, 9H).

Example 6: Synthesis of N-(6-methoxy-9-oxo-9H-fluoren-2-yl)pivalamide(Compound 1.006)

To a solution of compound 6.1 (2.0 g, 7.7 mmol, 1.0 eq) in toluene (20mL)/EtOH (5 mL)/H₂O (5 mL) was added compound 6.2 (1.29 g, 8.5 mmol, 1.1eq), Pd(PPh₃)₄ (92 mg, 0.8 mmol, 0.1 eq) and Na₂CO₃ (2.4 g, 23.1 mmol,3.0 eq) under nitrogen atmosphere. The mixture was stirred at 90° C. for2 h. Then the mixture was filtrated and extracted with EA and H₂O,separated and the organic layer was washed with brine, dried overNa₂SO₄, concentrated in vacuum. The residue was purified by columnchromatography on a silica gel (PE/EA, 20:1-15:1) to give compound 6.3(2.2 g, 100%) as a yellow oil. TLC: PE:EA=8:1, Rf_((6.1))=0.7,Rf_((6.3))=0.5.

To a solution of compound 6.3 (2.2 g, 7.7 mmol, 1.0 eq) in MeOH (20mL)/THF (20 mL) was added 2.5 M NaOH (6.2 mL, 15.4 mmol, 2.0 eq). Themixture was stirred at room temperature for 2 h. Then the mixture wasadded 1 M HCl to adjust pH=3, filtrated and dried in vacuum to givecompound 6.4 (1.78 g, 85%) as a white solid. TLC: PE:EA=1:3,Rf_((6.3))=1, Rf_((6.4))=0.1.

Compound 6.4 (1.7 g, 6.2 mmol, 1.0 eq) was added in PPA (30 mL), themixture was stirred at 120° C. for 4 h. Then the mixture was poured intoice water, filtrated and washed with H₂O and MeOH, then filtrated anddried in vacuum to give a mixture of compound 6.5a and 6.5b (1.4 g, 89%)as a yellow solid. TLC: PE:EA=1:3, Rf_((6.4))=0.1, Rf_((6.5))=0.8, 0.9.

To a solution of compound 6.5a and 6.5b (0.7 g, 2.7 mmol, 1.0 eq) inMeOH (30 mL)/THF (30 mL) was added Pd/C (70 mg, 10% wt). The resultingsolution was stirred at room temperature for 3 h under H2. The mixturewas filtrated and concentrated in vacuum to give a mixture of compound6.6a and 6.6b (0.57 g, 92%) as a brown solid. TLC: PE:EA=1:1,Rf_((6.5))=0.6, Rf_((6.6))=0.4.

To a solution of compound 6.6a and 6.6b (0.57 g, 2.5 mmol, 1.0 eq) indry THF (20 mL) was added Na₂CO₃ (1.06 g, 10.0 mmol, 4.0 eq) undernitrogen atmosphere, then pivaloyl chloride (1.5 g, 12.7 mmol, 5.0 eq)was added in. The mixture was stirred at room temperature for 0.5 h.Then the mixture was diluted with EA and H₂O, separated and the organiclayer was washed with saturated aqueous NaHCO₃ and brine, dried overNa₂SO₄, concentrated in vacuum. The residue was purified by columnchromatography on a silica gel (PE/EA, 6:1-2:1) to give a mixture ofcompound 6.7a and 6.7b (0.46 g, 56%) as a yellow solid. TLC: PE:EA=1:1,Rf_((6.6))=0.4, Rf_((6.7))=0.5.

To a solution of compound 6.7a and 6.7b (0.45 g, 1.45 mmol, 1.0 eq) inDCM (30 mL) was added PDC (1.6 g, 4.34 mmol, 3.0 eq) and SiO₂ (1 g). Themixture was stirred at room temperature for 2 h. Then the mixture wasfiltrated and concentrated in vacuum. The residue was purified by columnchromatography on a silica gel (PE/EA, 6:1-2:1) to give compound 6.8a(0.17 g, 38%) and 6.8b (0.28 g, 62%) as yellow solids. TLC: PE:EA=2:1,Rf_((6.7))=0.2, Rf_((6.8))=0.3

To a solution of compound 6.8a (100 mg, 0.32 mmol, 1.0 eq) in DCM (30mL) was added 2 M BBr₃ (1.6 mL, 3.2 mmol, 10.0 eq). The mixture wasstirred at room temperature for 0.5 h. Then the mixture was quenchedwith MeOH and extracted with DCM and H₂O, separated and the organiclayer was washed with saturated aqueous NaHCO₃ and brine, dried overNa₂SO₄, concentrated in vacuum. The residue was purified by prep-HPLC togive Compound 1.006 (51 mg, 41%) as a yellow solid. TLC: PE:EA=1:1,Rf_((6.8a))=0.5, Rf_((1.006))=0.8. LCMS: [M+1]+=296. ¹H NMR (400 MHz,CDCl₃): δ 8.31 (s, 1H), 7.78-7.75 (m, 1H), 7.59-7.58 (m, 1H), 7.40-7.38(m, 1H), 7.33 (s, 1H), 7.30-7.27 (m, 1H), 6.92-6.90 (m, 1H), 6.67-6.64(m, 1H), 1.27 (s, 9H).

Example 7: Synthesis of N-(7-hydroxy-9-oxo-9H-fluoren-2-yl)pivalamide(Compound 1.007)

A mixture of 7.1 (1.3 g, 5 mmol, 1.0 eq) and water (6 mL) was heated at110° C. HNO₃ (65%, 6 mL) and H2SO4 (96%, 9 mL) was then added dropwise.The mixture was heated at 110° C. for 6 h. Water was added and the crudeproduct was filtered, washed with water, and dried. The compound wastriturated with acetone to give compound 7.2 (1 g, 67%) as yellow solid.

To a mixture of 2 (100 mg, 0.32 mmol, 1.0 eq), iron powder (92 mg, 1.64mmol, 5.0 eq), NH₄Cl (175 mg, 3.28 mmol, 10.0 eq) in ethanol/water(v/v=2:1, 6 mL/3 mL) was stirred at 80° C. for 1 h under nitrogenatmosphere. After the reaction completely, the solid was filtered outand the filtrate was concentrated in vacuo. Then the residue waspurified by Prep-TLC (PE/EA, 1:1) to provide 7.3 (70 mg, 78%).

To a mixture of 7.3 (70 mg, 0.255 mmol, 1.0 eq) in THF (5 mL) was addedNa₂CO₃ (108 mg, 1.02 mmol, 4.0 eq) and 7.4 (62 mg, 0.51 mmol, 2.0 eq).The mixture was stirred at rt for 30 min under nitrogen atmosphere. Thenthe mixture was filtered, added H₂O (6 mL), extracted with EA (2×8 mL).The residue was dried over Na₂SO₄ and concentrated under reducedpressure. The residue was purified by Prep-TLC to give 7.5 (80 mg, 88%)as yellow solid.

A mixture of compound 7.5 (40 mg, 0.112 mmol, 1.0 eq), B₂(OH)₄ (50 mg,0.556 mmol, 5.0 eq), XPhosPdG2 (10 mg, 0.011 mmol, 0.1 eq), XPhos (12mg, 0.022 mmol, 0.2 eq), KOAc (54 mg, 0.556 mmol, 5.0 eq) in EtOH (6 mL)was degassed with N2 three times and heated to 80° C. for 3 h. Thereaction was monitored by TLC. Solvent was removed to provide crudecompound 7.6 (40 mg) as a yellow solid.

Compound 7.6 was dissolved in THF (5 mL) and acetic acid (0.4 mL) andtreated with hydrogen peroxide (1.6 mL). The reaction was stirred for 15min and then quenched with st. aq. NaHSO₃. The reaction was extractedwith EtOAc (2.×. 10 mL). The organic layers were combined, dried overNa₂SO₄, and concentrated in vacuo to a residue which was purified byPrep-TLC (PE/EA=1:1) to give Compound 1.007 (10.5 mg, 32%) as yellowsolid. LCMS: [M+1]=296. ¹H NMR (400 MHz, DMSO): δ 9.97 (s, 1H), 9.35 (s,1H), 7.88 (s, 1H), 7.77-7.15 (m, 1H), 7.53-7.47 (m, 2H), 6.94-6.90 (m,2H), 1.25-1.23 (m, 9H).

Example 8: Synthesis of N-(7-amino-9-oxo-9H-fluoren-2-yl)pivalamide(Compound 1.008)

To a mixture of compound 8.5 (5 g, 18.5 mmol, 1.0 eq) in EtOH (200 mL)was added Na₂S.9H₂O (20 g, 83.2 mmol, 4.5 eq) and NaOH (8 g, 200 mmol,10.8 eq) in H₂O (345 mL). The mixture was refluxed at rt for 5 h, thenstirred at 0° C. overnight. The reaction was monitored by TLC. Then themixture was filtered, washed with H₂O (2×50 mL), 5% NaOH (2×50 mL), H₂O(3×50 mL), cold EtOH (2×25 mL), ether (25 mL) and hexane (20 mL) to givecompound 8.6 (3.2 g, 82%).

To a mixture of compound 8.6 (200 mg, 0.952 mmol, 1.0 eq) in THF (10 mL)was added Na₂CO₃ (202 mg, 1.9 mmol, 2.0 eq) and compound 8.4 (114 mg,0.952 mmol, 1.0 eq). The mixture was stirred at −78° C. for 1 h. Thereaction was monitored by TLC. Then the mixture was quenched with water(10 mL). The precipitated solid was filtered, washed with THF (10 mL).The residue was purified by Prep-HPLC to give Compound 1.008 (5 mg, 4%)as a black solid. LCMS: [M+42]=336. ¹H NMR (400 MHz, DMSO-d₆): δ 9.30(s, 1H), 7.81 (s, 1H), 7.70 (d, J=8.0 Hz, 1H), 7.45-7.30 (m, 2H), 6.85(s, 1H), 6.73 (d, J=8.0 Hz, 1H), 1.22 (s, 9H).

Example 9: Synthesis of N-(9-oxo-9H-fluoren-2-yl)acetamide (Compound1.009)

To a mixture of 9.1 (50 mg, 0.26 mmol, 1.0 eq) in THF (3 mL) was addedNa₂CO₃ (83 mg, 0.78 mmol, 3.0 eq) and 9.2 (41 mg, 0.52 mmol, 2.0 eq).The mixture was stirred at rt for 30 min under nitrogen atmosphere. Thenthe mixture was filtered, added H₂O (5 mL), extracted with EA (3×5 mL).The residue was triturated with MeOH to give Compound 1.009 (35 mg, 58%)as red solid. LCMS: [M+1]=238. ¹H NMR (400 MHz, DMSO): δ 10.19 (s, 1H),7.92 (s, 1H), 7.70-7.65 (m, 3H), 7.57-7.54 (m, 2H), 7.31-7.27 (m, 1H),2.06 (s, 3H).

Example 10: Synthesis of 3,3-dimethyl-3,6-dihydro-2H-1,4-oxazine 4-oxide(Compound 1.010)

Compound 1.005 was prepared as described in Example 5. To a mixture ofCompound 1.005 (62 mg, 0.22 mmol) in methanol (3 mL) was added NaBH₄ (10mg, 0.26 mmol). After no starting material was observed in LC-MS and TLCanalysis, the reaction mixture was concentrated to remove methanol. Theresulting residue was purified by pTLC on silica gel to give 39 mgproduct (Compound 1.010), in 63% yield. TLC: hexane/ethyl acetate=3/1;R_(f) (starting material)=0.6; R_(f) (Compound 1.010)=0.2; LC-MS(ESI):282.4 [M+H]⁺; ¹H NMR (300 MHz, CDCl₃): δ 7.82 (s, 1H), 7.64-7.53 (m,4H), 7.34-7.26 (m, 2H), 5.49 (s, 1H), 1.32 (s, 9H).

Example 11: Synthesis of 1,1′-(9-oxo-9H-fluorene-2,7-diyl)diurea(Compound 1.011)

Compound 8.6 was prepared as described in Example 8. To a mixture ofcompound 8.6 (50 mg, 0.238 mmol, 1.0 eq) in HOAc/H₂O (5 mL/10 mL) wasadded Sodium cyanate (61.97 mg, 0.952 mmol, 4.0 eq) in H₂O (6 mL). Themixture was stirred at 50° C. for 2 h. The reaction was monitored byTLC. Then the mixture was quenched with water (5 mL). The precipitatedsolid was filtered, extracted with EA (20 mL). The residue was purifiedby Prep-HPLC to give Compound 1.013 (7 mg, 10%) as a brown solid. LCMS:[M+42]=338. ¹H NMR (400 MHz, DMSO): δ 8.74 (s, 2H), 7.71 (s, 2H),7.45-7.30 (m, 4H), 5.92 (s, 4H).

Example 12: Synthesis of N,N′-(9-oxo-9H-fluorene-2,7-diyl)diacetamide(Compound 1.012)

Compound 8.6 was prepared as described in Example 8. To a mixture ofcompound 8.6 (50 mg, 0.238 mmol, 1.0 eq) in THF (10 mL) was added Na₂CO₃(100.95 mg, 0.952 mmol, 4.0 eq) and AcCl (74.82 mg, 0.952 mmol, 4.0 eq).The mixture was stirred at rt for 10 min. The reaction was monitored byTLC. Then the mixture was quenched with water (10 mL). The precipitatedsolid was filtered, washed with THF (10 mL). The residue was purified byPre-HPLC to give Compound 1.012 (5 mg, 7%) as red solid. LCMS:[M+42]=336. ¹H NMR (400 MHz, DMSO): δ 10.17 (s, 2H), 7.90 (s, 2H),7.65-7.55 (m, 4H), 2.07 (s, 6H).

Example 13: Synthesis of N-(9-(hydroxyimino)-9H-fluoren-2-yl)pivalamide(Compound 1.013)

Compound 1.005 was prepared as described in Example 5. To a mixture ofCompound 1.005 (200 mg, 0.72 mmol, 1.0 eq) in EtOH (5 mL) was addedHONH₂.HCl (100 mg, 1.44 mmol, 2.0 eq). The mixture was refluxed at rtfor 16 h. The reaction was monitored by TLC. Then the mixture wasquenched with water (5 mL). The precipitated solid was filtered. Theresidue was dried over Na₂SO₄ and concentrated under reduced pressure.The residue was purified by Prep-TLC (PE:EA, 5:1) 4 times to giveCompound 1.013 (4 mg, 2%) as a yellow solid. TLC: PE:EA=2:1, UV 254 nm.Rf (Compound 1.013)=0.5. LCMS: [M+42]=336. ¹H NMR (400 MHz, CD₃OD): δ8.34 (d, J=8.0 Hz, 1H), 7.91 (s, 1H), 7.70-7.60 (m, 2H), 7.55-7.50 (m,1H), 7.42-7.36 (m, 1H), 7.28-7.24 (m, 1H), 1.31 (s, 9H).

Example 14: Synthesis of N-(3-hydroxy-9-oxo-9H-fluoren-2-yl)pivalamide(Compound 1.014)

Compound 14.1 (100 mg, 0.51 mmol, 1.0 eq) was dissolved in HOAc (2.0mL). Br₂ (100 mg, 0.61 mmol, 1.2 eq) was added dropwise at rt. Themixture was stirred at rt for 1 h. Water was added and the solid wasfiltered which was washed with water to give compound 14.2 (140 mg, 81%)as yellow solid.

To a mixture of 14.2 (140 mg, 0.42 mmol, 1.0 eq) in THF (3 mL) was addedNa₂CO₃ (134 mg, 1.26 mmol, 3.0 eq) and 14.3 (100 mg, 0.84 mmol, 2.0 eq).The mixture was stirred at rt for 30 min under nitrogen atmosphere. Thenthe mixture was filtered, added H₂O (5 mL), extracted with EA (3×5 mL).The residue was dried over Na₂SO₄ and concentrated under reducedpressure to give 14.4 (100 mg, 66%) as yellow solid.

A mixture of compound 14.4 (100 mg, 0.28 mmol, 1.0 eq), B₂(OH)₄ (125 mg,1.40 mmol, 5.0 eq), XPhosPdG2 (23 mg, 0.03 mmol, 0.1 eq), XPhos (29 mg,0.06 mmol, 0.2 eq), KOAc (137 mg, 1.40 mmol, 5.0 eq) in EtOH (10 mL) wasdegassed with N2 three times and heated to 80° C. for 6 h. The reactionwas monitored by TLC. Solvent was removed to give a crude yellowresidue. This crude oil was dissolved in THF (4 mL) and acetic acid (0.5mL) and treated with hydrogen peroxide (2 mL). The reaction was stirredfor 15 min and then quenched with st. aq. NaHSO₃. The reaction wasextracted with EtOAc (3×40 mL). The organic layers were combined, driedover Na₂SO₄, and concentrated in vacuo to a residue which was purifiedby Prep-HPLC to give Compound 1.014 (15 mg, 18%) as yellow solid. LCMS:[M+1]=296. ¹H NMR (400 MHz, DMSO): δ 8.56 (s, 1H), 8.12 (s, 1H),7.66-7.65 (m, 1H), 7.58-7.52 (m, 2H), 7.36-7.32 (m, 1H), 7.23 (s, 1H),1.27 (m, 9H).

Example 15: Synthesis of N-(9-amino-9H-fluoren-2-yl)pivalamide (Compound1.015)

Compound 1.005 was prepared as described in Example 5. To a mixture ofCompound 1.005 (50 mg, 0.18 mmol, 1.0 eq) in EtOH (3 mL) was addedHONH₂—HCl (100 mg, 1.44 mmol, 8.0 eq). The mixture was refluxed at rtovernight. Then the mixture was concentrated and dissolved in AcOH (6mL). The mixture was added Zn (120 mg, 1.85 mmol, 10.0 eq). The mixturewas refluxed at 80° C. for 2 h. The reaction was monitored by TLC. Thenthe mixture was filtered, dried over Na₂SO₄ and concentrated underreduced pressure. The residue was treated with EA and filtered to giveCompound 1.015 (14 mg, 28%) as a white solid in AcOH form. LCMS:[M+42]=322. ¹H NMR (400 MHz, DMSO-d₆): δ 9.28 (s, 1H), 7.98 (s, 1H),7.70-7.60 (m, 4H), 7.35-7.25 (m, 2H), 4.72 (s, 1H), 1.90 (s, 3H), 1.25(s, 9H).

Example 16: Synthesis of N-(6-hydroxy-9-oxo-9H-fluoren-2-yl)pivalamide(Compound 1.016)

Compound 6.8b was prepared as described in Example 6. To a solution ofcompound 6.8b (230 mg, 0.74 mmol, 1.0 eq) in DCM (30 mL) was added 2 MBBr₃ (3.7 mL, 7.4 mmol, 10.0 eq). The mixture was stirred at roomtemperature for 0.5 h. Then the mixture was quenched with MeOH andextracted with DCM and H₂O, separated and the organic layer was washedwith saturated aqueous NaHCO₃ and brine, dried over Na₂SO₄, concentratedin vacuum. The residue was purified by prep-HPLC to give Compound 1.016(4.7 mg, 5%) as a yellow solid. LCMS: [M+1]⁺=296. ¹H NMR (400 MHz,CD₃OD): δ 7.76-7.75 (m, 1H), 7.68-7.65 (m, 1H), 7.49-7.42 (m, 2H),6.95-6.94 (m, 1H), 6.61-6.58 (m, 1H), 1.29 (s, 9H).

Example 17: Synthesis of N-(9-hydroxy-9H-fluoren-2-yl)formamide(Compound 1.017)

To a mixture of compound 17.1 (100 mg, 0.513 mmol, 1.0 eq) in formicacid (3 mL) was added Ac₂O (3 drops). The mixture was stirred at roomtemperature for 0.5 h. The reaction was quenched by water and filtered.The filter cake was dissolved in EA and dried with Na₂SO₄. EA wasremoved to give compound 17.2 (105 mg, 92%) as light yellow solid.

To a mixture of compound 17.2 (105 mg, 0.471 mmol, 1.0 eq) in MeOH (10mL) was added NaBH₄ (54 mg, 1.41 mmol, 3.0 eq) at 0° C. The mixture wasstirred for 0.5 h. The mixture was extracted with EA and water. Theorganic layer was dried over Na₂SO₄ and concentrated under pressure togive a residue which was washed by MeOH to give Compound 1.017 (70 mg,66%) as white solid. LCMS: [M−1]−=224. ¹H NMR (400 MHz, DMSO): δ 10.23(s, 1H), 8.27 (s, 1H), 7.88 (s, 1H), 7.68-7.66 (m, 2H), 7.51 (d, J=7.6Hz, 1H), 7.36-7.18 (m, 2H), 5.81 (m, 1H), 5.41 (d, J=7.6 Hz, 1H)

Example 18: Synthesis of 2-(methylamino)-9H-fluoren-9-ol (Compound1.018)

To a mixture of compound 18.1 (1 g, 3.88 mmol, 1.0 eq), compound 18.2(520 mg, 7.76 mmol, 2.0 eq), Pd₂(dba)₃ (348 mg, 0.38 mmol, 0.1 eq),BINAP (486 mg, 0.78 mmol, 0.2 eq) and NaO^(t)Bu (1.49 g, 15.52 mmol, 4.0eq) in PhMe (10 mL) was refluxed at 100° C. for 16 h. The reaction wasmonitored by TLC. Then the mixture was diluted with H₂O (10 mL),extracted with EA (3×10 mL). The organic layer washed with brine. Theresidue was dried over Na₂SO₄ and concentrated under reduced pressure.The residue was purified by column chromatography on silica gel (PE/EA,5:1) to give compound 18.3 (500 mg, 62%). TLC: PE:EA=5:1, UV 254 nm. Rf(compound 18.1)=0.7. Rf (compound 18.3)=0.5.

To a mixture of compound 18.3 (500 mg, 2.39 mmol, 1.0 eq) in MeOH (5 mL)was added NaBH₄ (181 mg, 4.78 mmol, 2.0 eq) under nitrogen atmosphere.The mixture was stirred at rt for 2 h. The reaction was monitored byTLC. Then the mixture was quenched with H₂O, extracted with EA (2×10mL). The organic layer washed with brine. The residue was dried overNa₂SO₄ and concentrated under reduced pressure. The residue was purifiedby Prep-HPLC to give Compound 1.018 (150 mg, 30%) as a yellow solid.LCMS: [M+42]=253 ¹H NMR (400 MHz, d₆-DMSO): δ 7.64-7.60 (m, 2H),7.52-7.48 (m, 1H), 7.30-7.25 (m, 1H), 7.24-7.20 (m, 1H), 7.13 (s, 1H),6.93-6.89 (m, 1H), 5.40 (s, 1H), 2.83 (s, 3H).

Example 19: Synthesis of N-(3-oxo-2,3-dihydro-1H-inden-5-yl)acetamide(Compound 1.019)

To a solution of compound 19.1 (1 g, 5.6 mmol, 1.0 eq) in CH₃OH (20 mL)was added Pd/C (100 mg, 10% wt). The resulting solution was stirred atroom temperature for 14 hrs under H₂. The mixture was filtered to getfiltrate, removed in vacuo to give compound 19.2 (0.8 g, 96%) as a brownsolid, which was used directly in the next step without furtherpurification.

To a mixture of compound 19.2 (100 mg, 0.68 mmol, 1.0 eq) and TEA (206mg 2.04 mmol, 3.0 eq) in DMF (10 mL) was added compound 19.3 slowly at0° C. under N₂. The mixture was warmed to room temperature and stirredfor 14 hrs. The reaction mixture was poured into 50 ml water andextracted with EA (3×50 ml). The organic phase was washed with brine anddried over anhydrous Na₂SO₄. The mixture was concentrated under reducedpressure to get the residue, the residue was purified by columnchromatography on a silica gel (PE:EA=3:1) to obtain Compound 1.019 (70mg, 45%) as a white solid. TLC: PE:EA=3:1, R_(f) (Compound 1.019)=0.4,LC-MS: [M+MeCN+H]⁺=273.15. ¹H NMR (400 MHz, CDCl₃) δ 8.06 (dd, J=4.0 and8.0 Hz, 1H), 7.66 (d, J=4.0, 1H), 7.54 (s, 1H), 7.44 (d, J=8.0 Hz, 2H),3.10 (m, J=8.0 Hz, 2H)

Example 20: Synthesis of N-(9-ethoxy-9H-fluoren-2-yl)acetamide (Compound1.021) and N-(9-hydroxy-9H-fluoren-2-yl)acetamide (Compound 1.029)

To a mixture of compound 20.1 (100 mg, 0.42 mmol, 1.0 eq) intetrahydrofuran/methanol (3 mL/1 mL) was added Sodium borohydride (32mg, 0.84 mmol, 2.0 eq) at 0° C. The mixture was stirred at roomtemperature for 30 min under nitrogen atmosphere. The reaction wasmonitored by TLC. Then the mixture was added water (3 mL), ethyl acetate(3 mL) and filtered. The residue was dried over sodium sulfate andconcentrated under reduced pressure to obtain Compound 1.029 (61 mg,61%) as white solid. TLC: petroleum ether: ethyl acetate, 1:1, UV 254nm. R_(f): (compound 20.1)=0.5; R_(f): (Compound 1.029)=0.2. LCMS:[M−1]: 238. ¹H NMR (DMSO, 400 MHz): δ 9.84 (s, 1H), 7.89 (s, 1H),7.65-7.62 (m, 2H), 7.52-7.48 (t, J=8.0 Hz, 2H), 7.33-7.29 (t, J=7.4 Hz,1H), 7.23-7.20 (t, J=7.2 Hz, 1H), 5.79 (s, 1H), 5.39 (s, 1H) and2.04-2.03 (d, J=1.6 Hz, 3H).

The mixture of Compound 1.029 (80 mg, 0.33 mmol, 1.0 eq), Silver oxide(465 mg, 2.0 mmol, 6.0 eq) and Iodoethane (156 mg, 1.0 mmol, 3.0 eq) in1,2-Dichloroethane (5 mL) was stirred at 60° C. for 16 h. The reactionwas monitored by LCMS. Then the mixture was filtered. The residue wasdried over sodium sulfate and concentrated under reduced pressure. Theresidue was purified by prep-TLC to obtain Compound 1.021 (20 mg, 12%)as light yellow solid. LCMS: [M+1]: 268. ¹H NMR (DMSO, 400 MHz): δ 10.05(s, 1H), 7.89 (s, 1H), 7.70.7.68 (d, J=8.0 Hz, 2H), 7.57-7.52 (m, 2H),7.38-7.35 (t, J=7.2 Hz, 1H), 7.27-7.23 (t, J=7.4 Hz, 1H), 5.52 (s, 1H),3.36-3.32 (m, 2H), 2.05 (s, 3H) and 1.10-1.07 (t, J=7.0 Hz, 3H).

Example 21: Synthesis of 2-acetamido-9H-fluoren-9-yl acetate (Compound1.022)

The mixture of Compound 1.029 (50 mg, 0.21 mmol, 1.0 eq) and4-Dimethylaminopyridine (2.44 mg, 0.02 mmol, 0.1 eq) in aceticacid/acetic anhydride (1 mL/1 mL) was stirred at 70° C. for 16 h. Thereaction was monitored by LCMS. Then the mixture was filtered, addedwater (2 mL), extracted with ethyl acetate (3×2 mL). Then the mixturewas washed with methanol to obtain Compound 1.022 (8 mg, 14%) as whitesolid. LCMS: [M+23]: 304. ¹H NMR (DMSO, 400 MHz): δ 10.07 (s, 1H), 7.79(s, 1H), 7.73-7.71 (d, J=8.0 Hz, 2H), 7.65-7.63 (dd, J=8.4 Hz, 1.4 Hz,1H), 7.51-7.49 (d, J=7.2 Hz, 1H), 7.42-7.39 (t, J=7.6 Hz, 1H), 7.27-7.23(t, J=7.4 Hz, 1H), 6.66 (s, 1H), 2.14-2.12 (d, J=4.8 Hz, 3H) and 2.03(s, 3H).

Example 22: Synthesis of N-(9-ethoxy-9H-fluoren-2-yl)pivalamide(Compound 1.023)

Compound 1.010 was prepared as described in Example 10. The mixture ofCompound 1.010 (100 mg, 0.356 mmol, 1.0 eq), Silver oxide (247 mg, 1.068mmol, 3.0 eq) and Iodoethane (166 mg, 1.068 mmol, 3.0 eq) in1,2-Dichloroethane (10 mL) was stirred at 65° C. for 16 h. The reactionwas monitored by LCMS. Then the mixture was filtered. The residue wasdried over sodium sulfate and concentrated under reduced pressure. Theresidue was purified by prep-TLC to obtain Compound 1.023 (13 mg, 12%)as white solid. TLC: petroleum ether: ethyl acetate, 5:1, UV 254 nm.R_(f): (Compound 1.010)=0.1; R_(f): (Compound 1.023)=0.5. LCMS: [M+1]:310. ¹H NMR (DMSO, 400 MHz): δ 9.29 (s, 1H), 7.94 (s, 1H), 7.72-7.67 (m,3H), 7.54-7.53 (d, J=7.6 Hz, 1H), 7.38-7.34 (t, J=7.2 Hz, 1H), 7.27-7.23(td, J=7.4 Hz, 0.8 Hz, 1H), 5.51 (s, 1H), 3.42-3.35 (m, 2H), 1.23 (s,9H) and 1.18-1.07 (m, 3H).

Example 23: Synthesis of 2-pivalamido-9H-fluoren-9-yl acetate (Compound1.024)

Compound 1.010 was prepared as described in Example 10. The mixture ofCompound 1.010 (50 mg, 0.178 mmol, 1.0 eq) and 4-Dimethylaminopyridine(21.7 mg, 0.178 mmol, 1.0 eq) in acetic acid/acetic anhydride (3 mL/3mL) was stirred at 70° C. for 16 h. The reaction was monitored by TLC.Then the mixture was filtered, added water (5 mL), extracted with ethylacetate (3×5 mL). Then the mixture was washed with methanol to obtainCompound 1.024 (23 mg, 40%) as a white solid. TLC: petroleum ether:ethyl acetate, 5:1, UV 254 nm. R_(f): (Compound 1.010)=0.1; R_(f):(Compound 1.024)=0.4. LCMS: [M−1]: 322. ¹H NMR (DMSO, 400 MHz): δ 9.34(s, 1H), 7.86 (s, 1H), 7.74-7.73 (m, 3H), 7.51-7.49 (m, 1H), 7.41 (m,1H), 7.26 (m, 1H), 6.68 (s, 1H), 2.15 (s, 3H) and 1.22 (s, 9H).

Example 24: Synthesis of N-(9-methoxy-9H-fluoren-2-yl)pivalamide(Compound 1.025)

Compound 1.010 was prepared as described in Example 10. The mixture ofCompound 1.010 (50 mg, 0.178 mmol, 1.0 eq), Silver oxide (123.7 mg,0.534 mmol, 3.0 eq) and Iodomethane (38 mg, 0.267 mmol, 1.5 eq) in1,2-Dichloroethane (10 mL) was stirred at 40° C. for 16 h. The reactionwas monitored by LCMS. Then the mixture was filtered. The residue wasdried over sodium sulfate and concentrated under reduced pressure. Theresidue was purified by prep-TLC to obtain Compound 1.025 (6.6 mg,12.5%) as white solid. TLC: petroleum ether: ethyl acetate, 5:1, UV 254nm. R_(f): (Compound 1.010)=0.1; R_(f): (Compound 1.025)=0.5. LCMS:[M+23]: 318. ¹H NMR (DMSO, 400 MHz): δ 9.29 (s, 1H), 7.95 (m, 1H),7.72-7.70 (m, 3H), 7.54-7.53 (m, 1H), 7.38-7.36 (m, 1H), 7.28-7.27 (m,1H), 5.51 (s, 1H), 3.09 (s, 3H) and 1.30-1.23 (m, 9H).

Example 25: Synthesis of N-(9-cyano-9H-fluoren-2-yl)pivalamide (Compound1.026)

To the mixture of compound 21.1 (52.4 mg, 0.27 mmol, 1.5 eq) in ethanol(5 mL) was added tBuOK (30 mg, 0.27 mmol, 1.5 eq) and stirred at roomtemperature for 5 min. To the mixture was added Compound 1.005, preparedas described in Example 5, (50 mg, 0.18 mmol, 1.0 eq). The mixture wasstirred at room temperature for 2 h under nitrogen atmosphere. Thereaction was monitored by TLC. Then the mixture was filtered, addedwater (20 mL), extracted with ethyl acetate (3×20 mL). The organic layerwas washed with brine. The residue was dried over sodium sulfate andconcentrated under reduced pressure. The residue was purified byprep-TLC to obtain Compound 1.026 (20 mg, 38%) as white solid. ¹H NMR(CDCl₃, 400 MHz): δ 8.80 (s, 1H), 7.68-7.58 (m, 4H), 7.53-7.33 (m, 4H),4.56 (s, 1H) and 1.31 (s, 9H).

Example 26: Synthesis of 9-oxo-9H-fluorene-2,7-diyl diacetate (Compound1.027)

The mixture of compound 22.1 (50 mg, 0.236 mmol, 1.0 eq), Aceticanhydride (96.28 mg, 0.944 mmol, 4.0 eq) and 4-Dimethylaminopyridine(2.879 mg, 0.0236 mmol, 0.1 eq) in Pyridine (10 mL) was stirred at roomtemperature for 5 min. The reaction was monitored by LCMS. Then themixture was filtered, added water (5 mL), extracted with ethyl acetate(3×5 mL). Then the mixture was washed with 1N HCl. The residue waspurified by prep-HPLC to obtain Compound 1.027 (5.3 mg, 7.5%) as yellowsolid. LCMS: [M+42]: 338. ¹H NMR (DMSO, 400 MHz): δ 8.85-7.83 (m, 1H),7.41-7.42 (m, 2H), 7.39-7.37 (m, 2H) and 2.30 (s, 6H).

Example 27: Synthesis of N-(9-(hydroxyimino)-9H-fluoren-2-yl)pivalamide(Compound 1.028)

Compound 1.005 was prepared as described in Example 5. To a mixture ofCompound 1.005 (200 mg, 0.72 mmol, 1.0 eq) in ethanol (5 mL) was addedcompound 22.1 (100 mg, 1.44 mmol, 2.0 eq). The mixture was stirred atrefluxed for 16 h under nitrogen atmosphere. The reaction was monitoredby TLC. Then the mixture was dried over sodium sulfate and concentratedunder reduced pressure. The residue was purified by columnchromatography on a silica gel to obtain Compound 1.028 (150 mg, 71%) aswhite solid. TLC: petroleum ether: ethyl acetate, 2:1, UV 254 nm. R_(f):(Compound 1.005)=0.4; R_(f): (Compound 1.028)=0.2. LCMS: [M+42]: 336. ¹HNMR (CD₃OD, 400 MHz): δ 8.58 (s, 1H), 7.67-7.62 (m, 4H), 7.34-7.32 (m,1H), 7.24-7.22 (m, 1H), 4.56 (s, 1H) and 1.30 (s, 9H).

Example 28: Synthesis of 9-hydroxy-9H-fluoren-2-yl pivalate (Compound1.030)

A solution of compound 24.1 (100 mg, 0.51 mmol, 1.0 eq) and sodiumcarbonate (162 mg, 1.53 mmol, 3.0 eq) in THF (10 mL) was cooled to 0′Cand compound 24.2 (74 mg, 0.61 mmol, 1.2 eq) was added. The resultingmixture was stirred from 0° C. to room temperature overnight. Theprogress of the reaction mixture was monitored by TLC. After completionof the reaction, the mixture was filtered, diluted with water (1500 mL)and then extracted with dichloromethane (100 mL). The organic layer wasdried over anhydrous sodium sulfate and concentrated under reducedpressure. The residue was purified by prep-TLC (PE/EtOAc=10:1) to affordcompound 24.3 (105 mg, 73%) as a yellow solid.

To a solution of compound 24.3 (105 mg, 0.375 mmol, 1.0 eq) in methanol(5 mL) was added sodium borohydride (17 mg, 0.45 mmol, 1.2 eq) undernitrogen atmosphere. The resulting solution was stirred for 1 hour atroom temperature. The progress of the reaction mixture was monitored byTLC. After completion of the reaction, the mixture was concentratedunder reduced pressure and the residue was purified by prep-TLC(PE/EtOAc=10:1). The desired Compound 1.030 was obtained as a yellowsolid, 20.1 mg, in 19% yield. TLC: hexane/ethyl acetate (10:1). R_(f):(Compound 24.3)=0.5; R_(f): (Compound 1.030)=0.3; LC-MS: 281.00 [M−1].¹H NMR (400 MHz, CDCl₃): δ 7.64-7.57 (m, 3H), 7.40-7.27 (m, 3H),7.08-7.03 (m, 1H), 5.55 (s, 1H), 3.46 (s, 1H), 1.36 (s, 9H).

Example 29: Synthesis ofN-(9-hydroxy-9H-fluoren-2-yl)tetrahydro-2H-pyran-2-carboxamide (Compound1.031)

The mixture of compound 25.1 (160 mg, 1.23 mmol, 1.0 eq) in thionylchloride (5 mL) was refluxed at 85° C. for 2 h under nitrogenatmosphere. The reaction was monitored by TLC. Then the mixture wasdiluted with water, filtered, washed with water. The residue was driedover sodium sulfate and concentrated under reduced pressure to obtaincompound 25.2 (160 mg, crude), which was used directly in the next stepwithout further purification.

To a mixture of compound 25.3 (190 mg, 1.03 mmol, 1.0 eq) and sodiumcarbonate (436.72 mg, 4.12 mmol, 4.0 eq) in dry tetrahydrofuran (10 mL)was added compound 25.2 (160 mg, 1.23 mmol, 1.2 eq) at 0° C. The mixturewas stirred at room temperature overnight under nitrogen atmosphere. Thereaction was monitored by LCMS. Then the mixture was diluted with water,filtered, washed with water. The residue was dried over sodium sulfateand concentrated under reduced pressure to obtain compound 25.4 (260 mg,crude) as yellow solid. TLC: petroleum ether: ethyl acetate, 5:1, UV 254nm. R_(f): (compound 25.3)=0.5, R_(f): (compound 25.4)=0.45.

To a mixture of compound 25.4 (150 mg, 0.488 mmol, 1.0 eq) in methanol(5 mL) was added sodium borohydride (92.32 mg, 2.44 mmol, 4.5 eq) at 0°C. The mixture was stirred at room temperature for 5 min under nitrogenatmosphere. The reaction was monitored by TLC. To the mixture was addedwater, which was then filtered and washed with water. The residue wasdried over sodium sulfate and concentrated under reduced pressure. Theresidue was purified by prep-HPLC to obtain Compound 1.031 (73 mg, 48%)as white solid. TLC: petroleum ether: ethyl acetate, 5:1, UV 254 nm.R_(f): (compound 25.4)=0.6; R_(f): (Compound 1.031)=0.2. LCMS: [M+1]:310. ¹H NMR (DMSO, 400 MHz): δ 9.57 (s, 1H), 8.02 (s, 1H), 7.63 (m, 2H),7.57 (m, 1H), 7.50 (m, 1H), 7.33-7.29 (m, 1H), 7.24-7.20 (m, 1H), 5.40(s, 1H), 4.02-3.99 (m, 2H), 3.52-3.46 (m, 1H), 1.91-1.81 (m, 2H) and1.56-1.43 (m, 4H).

Example 30: Synthesis of4-hydroxy-N-(9-hydroxy-9H-fluoren-2-yl)benzamide (Compound 1.032)

The mixture of compound 26.1 (216 mg, 1.2 mmol, 1.0 eq) in thionylchloride (2 mL) was stirred at 80° C. for 1 h under nitrogen atmosphere.The reaction was monitored by TLC. Then the mixture was quenched withmethanol. The residue was dried over sodium sulfate and concentratedunder reduced pressure to obtain compound 26.2 (245 mg, crude) as alight yellow oil, which was used directly in the next step withoutfurther purification.

To a mixture of compound 26.3 (195 mg, 1.0 mmol, 1.0 eq) and sodiumcarbonate (530 mg, 5.0 mmol, 5.0 eq) in dry tetrahydrofuran (5 mL) wasadded compound 26.2 (238.2 mg, 1.2 mmol, 1.2 eq) at 0° C. The mixturewas stirred at room temperature for 2 h under nitrogen atmosphere. Thereaction was monitored by LCMS. Then the mixture was filtered and thefiltrate was concentrated under reduced pressure to obtain compound 26.4(530 mg, crude) as light yellow solid, which was used directly in thenext step without further purification.

To a solution of compound 26.4 (530 mg, 1.0 mmol, 1.0 eq) intetrahydrofuran (5 mL) was added potassium carbonate (276 mg, 2.0 mmol,2.0 eq). The mixture was stirred at room temperature overnight undernitrogen atmosphere. The reaction was monitored by LCMS. Then thesuspension was filtered and the filtrate was concentrated under reducedpressure to obtain compound 26.5 (400 mg, crude) as red solid, which wasused directly in the next step without further purification.

To a mixture of compound 26.5 (400 mg, 1.27 mmol, 1.0 eq) in methanol (5mL) was added Sodium borohydride (129 mg, 3.81 mmol, 3.0 eq) at 0° C.The mixture was stirred at room temperature for 4 h under nitrogenatmosphere. The reaction was monitored by TLC. The solution was purifiedby acid prep-HPLC to obtain Compound 1.032 (86.7 mg, 21%) as whitesolid. LCMS: [M+1]: 318. ¹H NMR (DMSO, 400 MHz): δ 10.07-10.05 (d, J=9.2Hz, 2H), 8.06 (s, 1H), 7.84 (m, 2H), 7.60 (m, 3H), 7.52 (m, 1H), 7.31(m, 1H), 7.21 (m, 1H), 6.85-6.83 (d, J=8.4 Hz, 2H), 5.82-5.80 (d, J=7.6Hz, 1H) and 5.44-5.42 (d, J=7.6 Hz, 1H).

Example 31: Synthesis of 3-(9-hydroxy-9H-fluoren-2-yl)-1,1-dimethylurea(Compound 1.033)

Compound 27.1 (200 mg, 1.026 mmol, 1.0 eq), compound 27.2 (220 mg, 2.05mmol, 2.0 eq), 4-Dimethylaminopyridine (125 mg, 1.02 mmol, 1.0 eq) andpyridine (324 mg, 4.1 mmol, 4.0 eq) was sequentially added under air toa reaction tube equipped with a stir bar and a septum. Dichloromethane(10 mL) was added by syringe the resulting mixture vigorously stirredfor 24 h at ambient temperature. After this time, the contents of theflask were extracted with ethyl acetate. The solution obtained wasfiltered through the plug of silica gel and anhydrous Magnesium sulfate,and then concentrated by rotary evaporation. The residue was purified byflash chromatography, eluting with hexane/ethyl acetate to affordcompound 27.3 (150 mg, 55%). TLC: petroleum ether: ethyl acetate, 2:1,UV 254 nm R_(f): (compound 27.1)=0.5; R_(f): (compound 27.3)=0.2.

To a mixture of compound 27.3 (120 mg, 0.45 mmol, 1.0 eq) in methanol (5mL) was added Sodium borohydride (68.6 mg, 1.8 mmol, 4.0 eq) at 0° C.The mixture was stirred at room temperature for 1 h under nitrogenatmosphere. The reaction was monitored by TLC. Then the mixture wasadded water, filtered and washed with water. The residue was dried oversodium sulfate and concentrated under reduced pressure. The residue waswashed with methanol to obtain Compound 1.033 (46 mg, 81%) as whitesolid. TLC: petroleum ether: ethyl acetate, 3:1, UV 254 nm. R_(f):(compound 27.3)=0.5; R_(f): (Compound 1.033)=0.3. LCMS: [M+1]: 269. ¹HNMR (d₆-DMSO, 400 MHz): δ 8.33 (s, 1H), 7.75 (s, 1H), 7.55 (m, 2H),7.50-7.48 (d, J=7.2 Hz, 1H), 7.44-7.43 (d, J=2.0 Hz, 1H), 7.42-7.41 (d,J=1.6 Hz, 1H), 7.29-7.27 (m, 1H), 7.21-7.19 (m, 1H), 5.74-5.72 (d, J=7.6Hz, 1H), 5.38-5.36 (d, J=7.6 Hz, 1H) and 2.91 (s, 6H).

Example 32: Synthesis of2,2,2-trichloro-N-(9-hydroxy-9H-fluoren-2-yl)acetamide (Compound 1.034)

To a mixture of compound 27.1 (150 mg, 0.77 mmol, 1.0 eq) and sodiumcarbonate (326 mg, 3.08 mmol, 4.0 eq) in dry tetrahydrofuran (6 mL) wasadded compound 28.1 (277 mg, 1.54 mmol, 2.0 eq) at 0° C. The mixture wasstirred at room temperature for 10 min under nitrogen atmosphere. Thereaction was monitored by TLC. Then the mixture was diluted with water,filtered, washed with water. The residue was dried over sodium sulfateand concentrated under reduced pressure to obtain compound 28.2 (150 mg,58%) as white solid. TLC: petroleum ether: ethyl acetate, 2:1, UV 254nm, R_(f): (compound 27.1)=0.4; R_(f): (compound 28.2)=0.6.

To a mixture of compound 28.2 (150 mg, 0.44 mmol, 1.0 eq) in methanol (5mL) was added sodium borohydride (68 mg, 1.76 mmol, 4.0 eq). The mixturewas stirred at room temperature for 1 h under nitrogen atmosphere. Thereaction was monitored by TLC. Then the mixture was quenched with satammonium chloride, diluted with water, filtered and washed with water.The residue was dried over sodium sulfate and concentrated under reducedpressure to obtain Compound 1.034 (45 mg, 30%) as white solid. LCMS:[M+1]: 343. ¹H NMR (CDCl₃, 400 MHz): δ 10.86 (s, 1H), 7.91 (s, 1H),7.77-7.71 (dd, J=16.0 Hz 8.0 Hz, 2H), 7.62-7.60 (d, J=8.0 Hz, 1H),7.55-7.54 (d, J=7.6 Hz, 1H), 7.35-7.33 (m, 1H), 7.29-7.27 (m, 1H),5.88-5.86 (d, J=7.6 Hz, 1H) and 5.47-5.45 (d, J=7.6 Hz, 1H).

Example 33: Isolation and Enhancement of Hematopoietic Stem CellsDerived from Non-Mobilized Peripheral Blood Using Compounds of Formula I

This Example demonstrates the enhancement of HSCs in cultures withcompounds of Formula I.

Materials and Methods

CD34+ cells were isolated from donor peripheral blood. Standard buffycoat separation using ficoll paque was performed. Cells were pelletedand incubated with unlabeled CD64 antibody. Cells then underwentnegative depletion using biotinylated CD2, CD3, CD4, CD5, CD8, CD11b,CD14, CD16, CD19, CD20, CD45RA, CD56, CD235 (in some examples CD15, CD25and other lineage specific antibodies may also be used). Cells whichbind these antibodies are depleted using streptavidin beads. Theenriched progenitor pool then undergoes cell sorting for CD34+.

Isolated CD34+ cells were incubated in an in vitro culture media ofAlpha MEM without phenol red with 10% (v/v) heat inactivated fetalbovine serum (FBS). When testing compounds of Formula I, two internalcontrols were used: a positive control (+SF conditions) and a baselinecontrol (i.e. basic conditions (“cytokines only”)). The media componentsand concentrations used for the compounds tested are described in Table2. The culture also included an antibiotic solution that includespenicillin, streptomycin, and amphotericin B to avoid contamination.Controls were included because the amount of expansion in samplesobtained varies from individual to individual.

TABLE 2 Additional Components included in the culture media of BasicConditions (cytokines only); positive control (+SF conditions); and+Formula I conditions (conditions where a compound of Formula I isadded). - Factor - - Concentration - Cytokines/Growth Factors BaseConditions TPO 100 ng/mL (Cytokines Only) SCF 100 ng/mL FLT3L 100 ng/mLCytokines/Growth Factors +SF Conditions TPO 100 ng/mL (Positive Control)SCF 100 ng/mL FLT3L 100 ng/mL Small Molecules SF1670 500 nMCytokines/Growth Factors +Formula I TPO 100 ng/mL Conditions SCF 100ng/mL FLT3L 100 ng/mL Small Molecules Compound of 63 nM, 125 nM, 250 nM,Formula I 500 nM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM or as indicated in FIGS.1-23.

Cultures were incubated at 3% oxygen (controlled by nitrogen) and 5%CO₂.

Small molecule components were added separately and fresh each time themedia needs to be refreshed. Cytokines can be stored together. Mediarenewal should occur at least every few days.

On the days indicated one-half of the volume of the cell culture wasremoved for data analysis (flow cytometry using a BD FACS ARIA II). Theculture volume was replenished with fresh media according to theconditions tested. The data reported accounts for the dilution factorintroduced in this procedure.

Separate experiments were performed for each compound tested (Compound1.001-1.023).

Results

The expansive effect of Compounds 1.001 to 1.023 are displayed in FIG.1-FIG. 23. The graphs in each figure report the fold change in cellsbetween days 2 and 7. Each column in the figures report the fold changein cells at the noted concentration of compound of Formula I tested. Thethin dashed line reports the expansive effect of the basic conditions(i.e. cytokines only), and thick dashed line reports the expansiveeffect of the +SF conditions (500 nM SF 1670). Collectively, these datademonstrate that compounds of Formula I provide a positive expansiveeffect of HSCs in culture.

Table 3, below, summarizes the relative expansive effect of Compound1.001 to 1.023 (sample compounds) at the indicated concentration. Thedata in Table 3 is reported as the relative expansive effect. Therelative expansive effect is a normalized value of the fold changesshown in each of FIG. 1-FIG. 23. It is calculated as shown below:

$\frac{\begin{matrix}{{{Sample}{Compound}{Fold}{change}} -} \\{{Basic}{Conditions}{Fold}{Change}}\end{matrix}}{\begin{matrix}{{{+ {SF}}{Conditions}{Fold}{Change}} -} \\{{Basic}{Conditions}{Fold}{Change}}\end{matrix}} = {{Relative}{Fold}{Change}}$

TABLE 3 Relative expansive effect of treatment with compounds of FormulaI on CD34+/CD133+ cells (“CD133 effect”) and CD34+/CD133+/CD90+ cells(“CD90 effect”) in cultures containing Compounds 1.001-1.023 (samplecompounds) at the indicated concentrations. Concentration of sampleCD133 CD90 Compound compound (μM) effect effect 1.001 0.5 + ++ 1.00216 + ++ 1.003 0.5 ++ ++ 1.004 8 ++ +++ 1.005 0.125 ++ +++ 1.006 1 +++++++ 1.007 4 +++ ++++ 1.008 4 +++ +++++ 1.009 2 +++++ +++++ 1.010 16+++++ +++++ 1.011 0.125 + ++ 1.012 0.25 ++ ++ 1.013 4 +++ +++ 1.014 8 +++++ 1.015 2 +++ ++++ 1.016 16 ++ ++++ 1.017 4 + ++ 1.018 4 + ++ 1.019 8++ ++ 1.020 16 ++ ++ 1.021 8 ++ ++ 1.022 32 +++ +++++ 1.023 32 + ++

The reported values (e.g., +, ++, and +++) for relative expansive effectof compounds of Formula I on CD34+/CD133+ and CD34+/CD133+/CD90+ cellspresented in Table 3 are shown below, where “x” is the calculatedrelative fold-change.

Relative Fold Change Value x < 0.2 + 0.2 ≤ x < 0.55 ++ 0.55 ≤ x < 0.9+++ 0.9 ≤ x < 1.25 ++++ 1.25 ≤ x +++++

Example 34: Enhancement of Hematopoietic Stem Cells Derived from CordBlood in Culture Using a Compound of Formula I

This Example describes the culturing of hematopoietic stem cells derivedfrom cord blood when cultured in the presence of Compound 1.008. Thenumber of HSCs in culture continues to increase through 19 days of invitro incubation.

Materials and Methods

A frozen cord blood sample was thawed and gradually brought to roomtemperature. Thawed cord blood was incubated in an in vitro culturemedia of Alpha MEM without phenol red, 10% (v/v) heat inactivated fetalbovine serum (FBS). Four samples were tested: Base conditions, +SFConditions, +1.008 Conditions, and +1.008/+ER conditions. The componentsincluded in each condition is described in Table 4. Each conditiontested also included an antibiotic solution that includes penicillin,streptomycin, and amphotericin B to avoid contamination.

TABLE 4 Additional Components included in the culture media of BaseConditions, +SF Conditions, +1.008 Conditions (with Compound 1.008),+1.008/+ER conditions. - Factor - - Concentration - Cytokines/GrowthFactors Base TPO 100 ng/mL Conditions SCF 100 ng/mL FLT3L 100 ng/mLCytokines/Growth Factors +SF TPO 100 ng/mL Conditions SCF 100 ng/mLFLT3L 100 ng/mL Small Molecules SF1670 500 nM Cytokines/Growth Factors+1.008/+ER TPO 100 ng/mL Conditions SCF 100 ng/mL FLT3L 100 ng/mL SmallMolecules Compound 1.008 250 nM Cytokines/Growth Factors +1.008/+ER TPO100 ng/mL Conditions SCF 100 ng/mL FLT3L 100 ng/mL Small MoleculesER50891 (RAR 100 nM receptor antagonist) Compound 1.008 250 nM

Cultures were incubated at 3% oxygen (controlled by nitrogen) and 5%CO₂.

Small molecule components were added separately and fresh each time themedia needs to be refreshed. Cytokines can be stored together. Mediarenewal should occur at least every few days.

On the days indicated varying amounts of the cell culture was removedfor data analysis (flow cytometry using a BD FACS ARIA II) and to avoidovercrowding of cells. The culture volume was replenished with freshmedia according to the conditions tested. The data reported accounts forthe dilution factor introduced in this procedure.

Results

Flow cytometric analysis of +1.008 Conditions demonstrates thathematopoietic stem cells are maintained and continue to expand evenafter 19 in culture (FIG. 24A-E). In fact, FIG. 25A-4E shows that after19 days in culture there is a greater than 50-fold increase in CD34+cells (FIG. 25B) and CD34+/CD133+ cells (FIG. 25C) from day 2, about a20-fold increase in CD34+/CD133+/CD90+ cells (FIG. 25D) from day 2, andover a 12-fold increase in CD34+/CD133+/CD90+/CD38^(low/−) cells (FIG.25E) from day 2 in cord blood samples cultured in the presence of+1.008. These levels are even further improved with the addition ofER50891.

Example 35: Enhancement of Hematopoietic Stem Cells Derived from CordBlood Using Compounds of Formula I

Materials and Methods

CD34+ cells from cord blood were purchased from STEMCELL Technologies.Primary human CD34+ cells were isolated from cord blood samples usingpositive immunomagnetic separation techniques. Cells were thawed andgradually brought to room temperature. Samples were washed, then placedin overnight culture in StemSpan with 100 ng/ml each of FLT3L, TPO, SCF,and IL-6. Eighteen to twenty-four hours later (day 1), cells werecounted and immunophenotyped (flow cytometry on an Invitrogen Attune NxTcytometer).

Approximately 1000 live cells were plated into each well of 96-wellplates; exact cell numbers dispensed per well were quantified with flowcytometry for later calculations.

Media for testing compounds of Formula I was prepared using Alpha MEMwithout phenol red, 10% (v/v) heat inactivated fetal bovine serum.Culture conditions also included an antibiotic solution that includespenicillin, streptomycin, and amphotericin B to avoid contamination.Additional media components and concentrations used for the compoundstested are described in Table 5.

TABLE 5 Additional Components included in the culture media of BaseConditions (cytokines only), +Formula I conditions. - Factor - -Concentration - Cytokines/Growth Factors Base Conditions TPO 100 ng/ml(Cytokines Only) SCF 100 ng/ml FLT3L 100 ng/ml IL-6 100 ng/mlCytokines/Growth Factors +Formula I TPO 100 ng/ml Conditions SCF 100ng/ml FLT3L 100 ng/ml IL-6 100 ng/ml Small Molecules Compound ofConcentrations Formula I tested are indicated in FIGS. 26-51 and in theparagraph below.

Compounds 1.005, 1.006, 1.007, 1.008, 1.009, 1.010, 1.013, 1.014, 1.015,1.021, 1.022, 1.023, 1.024, 1.025, 1.026, 1.027, 1.028, and 1.029 weretested in duplicate wells at 0.5, 2, and 8 μM. Compounds 1.030-1.035were tested in triplicate wells at 0.1, 0.316, 1.0, 3.16, and 10 μM.Compound 1.036 was tested in duplicate wells at 0.149, 0.310, 0.647,1.351, 2.819, and 10 μM. Compound 1.037 was tested in single wells at0.253, 0.527, 1.100, 2.296, 4.792, and 10 μM.

All incubations for this experiment took place at 3% oxygen (controlledby nitrogen) and 5% CO₂. Following seven days of culture, cells fromwells were collected and phenotypes were analyzed (flow cytometry on anInvitrogen Attune NxT cytometer).

Results

The expansive effects for compounds 1.005, 1.006, 1.007, 1.008, 1.009,1.010, 1.013, 1.014, 1.015, 1.021, 1.022, 1.023, 1.024, 1.025, 1.026,1.027, 1.028, 1.029, 1.030, 1.031, 1.032, 1.033, 1.034, 1.035, 1.036,and 1.037 are displayed in FIG. 26-FIG. 51.

The graphs in each figure report the fold change in cells between days 1and 7. Each point in the figures reports the average fold change of theindicated number of replicates at the noted concentration of thecompound of Formula I tested. Error bars display the maximum and minimumfold change measured at that concentration. The dashed line reports theexpansive effect of the base conditions (i.e. cytokines only).Collectively, these data demonstrate that treatment with compounds ofFormula I provides a positive expansive effect to cord blood-derivedHSCs in culture.

Table 6 below, summarizes the relative expansive effect of the screenedcompounds at the indicated concentration. The data in Table 6 isreported as the relative expansive effect. The relative expansive effectis a normalized value of the fold changes shown in each of the figures.It is calculated as shown below:

$\frac{{Sample}{compound}{fold}{change}}{{Base}{conditions}{fold}{change}} = {{Relative}{Fold}{Change}}$

TABLE 6 Relative expansive effect of treatment with compounds of FormulaI on CD34+/CD133+ cells (“CD133 effect”) and CD34+/CD133+/CD90+ cells(“CD90 effect”) in cultures containing the indicated compounds at theindicated concentrations. Concentration of sample CD133 CD90 Compoundcompound (μM) effect effect 1.005 0.5 ++ ++ 1.006 2 ++ ++ 1.007 2 ++++++++ 1.008 8 ++++ ++++ 1.009 0.5 +++ +++ 1.010 8 +++++ +++++ 1.013 2++++ +++++ 1.014 8 ++ ++ 1.015 0.5 ++ ++ 1.021 8 + + 1.022 8 +++ ++1.023 8 ++ ++ 1.024 8 +++ ++++ 1.025 8 +++ ++++ 1.026 2 ++ ++ 1.027 2 ++++ 1.028 2 +++ +++ 1.029 8 ++++ ++++ 1.030 10 +++ +++ 1.031 10 +++ ++1.032 10 ++ ++ 1.033 10 +++ ++ 1.034 10 +++++ +++++ 1.035 10 +++ +++1.036 10 ++ +++ 1.037 10 + ++

The reported values (e.g., +, ++, and +++) for relative expansive effectof compounds of Formula I on CD34+/CD133+ and CD34+/CD133+/CD90+ cellspresented in Table 6 are shown below, where “x” is the calculatedrelative fold-change.

Relative Fold Change Value x < 1.44 + 1.44 ≤ x < 1.8 ++ 1.8 ≤ x < 2.16+++ 2.16 ≤ x < 2.52 ++++ 2.52 ≤ x +++++

Example 36: Long-Term Enhancement of Hematopoietic Stem Cells Derivedfrom Mobilized Peripheral Blood, Non-Mobilized Peripheral Blood, andCord Blood, Using a Compound of Formula I

This examples demonstrates the enhancement and expansion ofhematopoietic stem cells for 21 days in culture using HSCs derived fromvarious sources.

Materials and Methods

CD34+ cells from mobilized peripheral blood were purchased from STEMCELLTechnologies. The blood from volunteer donors was mobilized using G-CSF.Volunteers were administered a maximum of 10 μg/kg/day of granulocytecolony-stimulating factor (G-CSF) for 3-5 days prior to collection.Primary human CD34+ cells were isolated from mobilized peripheral bloodleukapheresis samples using positive immunomagnetic separationtechniques.

CD34+ cells from cord blood were purchased from STEMCELL Technologies.Primary human CD34+ cells were isolated from cord blood samples usingpositive immunomagnetic separation techniques.

CD34+ cells from non-mobilized peripheral blood were purchased fromSTEMCELL Technologies. Primary human CD34+ cells were isolated fromblood samples using positive immunomagnetic separation techniques.

Cryspreserved CD34+ cell samples from each source were thawed andgradually brought to room temperature. Samples were washed then placedin overnight culture in StemSpan with 100 ng/ml each of FLT3L, TPO, SCF,and IL-6.

Cultures were incubated at 3% oxygen (controlled by nitrogen) and 5%CO₂.

Twenty-four hours later (day 1), cells were counted and immunophenotyped(flow cytometry on an Invitrogen Attune NxT cytometer). The mediacomponents and concentrations used for the compounds tested aredescribed in Table 7. Culture conditions also included an antibioticsolution that includes penicillin, streptomycin, and amphotericin B toavoid contamination. Approximately 1000 cells were added to each of thecord blood and mobilized peripheral blood flasks (5 ml total volume) orwells of a 96-well plate (200 μl total volume). Approximately 2000 cellswere added to each of the non-mobilized peripheral blood flasks (5 mltotal volume) or wells of a 96-well plate (200 μl total volume). Exactcell numbers dispensed per condition were quantified for latercalculations of fold change from day 1.

Wells were analyzed at days 7 and 10, flasks were analyzed at 14 and 21days of incubation. Cell numbers and phenotypes were quantified withflow cytometry. At day 14, fresh conditions for flasks were prepared ason day 1, and cells were split 1:20 into the new flasks. An additionalseven days later (day 21 of culture), cell numbers and phenotypes wereagain quantified with flow cytometry. Cell numbers calculated at day 21account for the passaging of the cells.

TABLE 7 Media Components included in the Base Conditions (cytokinesonly), +1.010 conditions. - Factor - - Concentration - Cytokines/GrowthFactors Base Conditions TPO 100 ng/ml (Cytokines Only) SCF 100 ng/mlFLT3L 100 ng/ml IL-6 100 ng/ml Small Molecules Vehicle control 0.05% v/v(DMSO) Cytokines/Growth Factors +1.010 Condition TPO 100 ng/ml SCF 100ng/ml FLT3L 100 ng/ml IL-6 100 ng/ml Small Molecules Compound 1.010 8 μM

Conditions in 96-well plates were prepared in quadruplicate; conditionsin flasks were prepared in duplicate. On the above-indicated number ofdays in culture, cells from wells or flasks were collected andphenotypes were analyzed (flow cytometry on an Invitrogen Attune NxTcytometer).

Results

Flow cytometric analysis of +1.010 Condition demonstrates thathematopoietic stem cells from diverse sources are maintained andcontinue to expand up to 21 days in culture (see, FIG. 52-FIG. 54). Infact, FIG. 52 shows that in cord blood, there is a greater than 300-foldexpansion of CD34+ cells (FIG. 52B), a greater than 600-fold expansionof CD34+/CD133+ cells (FIG. 52C), a greater than 1000-fold expansion ofCD34+/CD133+/CD90+ cells (FIG. 52D), a greater than 1500-fold expansionof CD34+/CD133+/CD90+/CD38^(low/−) cells (FIG. 52E) and a greater than200-fold expansion of CD34+/CD133+/CD90+/CD45RA− cells (FIG. 52F). Inmobilized peripheral blood (FIG. 53), there is a greater than 20-foldexpansion of CD34+ cells (FIG. 53B), a greater than 40-fold expansion ofCD34+/CD133+ cells (FIG. 53C), a greater than 60-fold expansion ofCD34+/CD133+/CD90+ cells (FIG. 53D), a greater than 60-fold expansion ofCD34+/CD133+/CD90+/CD38^(low/−) cells (FIG. 53E) and a greater than30-fold expansion of CD34+/CD133+/CD90+/CD45RA− cells (FIG. 53F). Innon-mobilized peripheral blood (FIG. 54), there is a greater thannine-fold expansion of CD34+ cells (FIG. 54B), a greater than 40-foldexpansion of CD133+ cells (FIG. 54C), and a greater than 60-foldexpansion of CD90+ cells (FIG. 54D), a greater than 200-fold expansionof CD34+/CD133+/CD90+/CD38^(low/−) cells (FIG. 54E) and a greater than30-fold expansion of CD34+/CD133+/CD90+/CD45RA− cells (FIG. 54F). In allcases, expansion with Compound 1.010 far surpasses expansion withcytokines alone.

Example 37: Enhancement of Hematopoietic Stem Cells at Atmospheric 02Using a Compound of Formula I

This examples demonstrates the enhancement and expansion ofhematopoietic stem cells at atmospheric oxygen using a compounds ofFormula I.

Materials and Methods

CD34+ cells from cord blood were purchased from STEMCELL Technologies.Primary human CD34+ cells were isolated from cord blood samples usingpositive immunomagnetic separation techniques. Cells were thawed andgradually brought to room temperature. Samples were washed, then placedin overnight culture in StemSpan SFEM with 100 ng/ml each of FLT3L, TPO,SCF, and IL-6.

Cultures were incubated at atmospheric oxygen (approximately 20%) and 5%CO₂.

Eighteen hours later (day 1), cells were counted and immunophenotyped(flow cytometry on an Invitrogen Attune NxT cytometer). The media wasprepared using StemSpan SFEM, with additional components andconcentrations used for the compounds tested described in Table 8.Culture conditions also included an antibiotic solution that includespenicillin, streptomycin, and amphotericin B to avoid contamination.Five mL of the respective conditions were added to 25 cm² flasks.Approximately 1000 cells were added to each flask; exact cell numbersper flask were quantified for later calculations of fold change from day1.

At nine days of incubation, cell numbers and phenotypes were quantifiedwith flow cytometry.

TABLE 8 Additional Components included in the culture media of BaseConditions (cytokines only), +Compound 1.010 conditions. - Factor - -Concentration - Cytokines/Growth Factors Base Conditions TPO 100 ng/ml(Cytokines Only) SCF 100 ng/ml FLT3L 100 ng/ml IL-6 100 ng/ml SmallMolecules Vehicle control 0.01% v/v (DMSO) Cytokines/Growth Factors+1.010 Condition TPO 100 ng/ml SCF 100 ng/ml FLT3L 100 ng/ml IL-6 100ng/ml Small Molecules Compound 1.010 8 μM

Conditions were prepared in duplicate.

Results

Flow cytometric analysis of the +1.010 Condition demonstrates thatCompound 1.010 has a positive expansive effect on hematopoietic stemcells when cultured for nine days under atmospheric oxygen. In fact,FIG. 55 shows a more than 150-fold expansion of CD34+ cells (FIG. 55B),and a more than 200-fold expansion of both CD34+/CD133+(FIG. 55C) andCD34+/CD133+/CD90+ cells (FIG. 55D).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference. Where a conflictexists between the instant application and a reference provided herein,the instant application shall dominate.

What is claimed is:
 1. A compound of Formula IIIa1

or a pharmaceutically acceptable salt, hydrate, or solvate thereof;wherein, R¹ is —NH—C(O)—R^(1a) NR^(b)—C(O)—R^(1a), R^(1a) is selectedfrom the group consisting of C₂₋₆ alkyl and C₂₋₆ haloalkyl; R² is —OH;and the subscript n is 0 or
 1. 2. The compound of claim 1, wherein n is0.
 3. The compound of claim 1, wherein n is
 1. 4. The compound of claim1, wherein R^(1a) is C₂₋₆ haloalkyl.
 5. The compound of claim 1, whereinR^(1a) is C₂₋₆ alkyl.
 6. The compound of claim 1, wherein n is 0 andR^(1a) is C₂₋₄ alkyl.
 7. The compound of claim 1, wherein n is 0 andR^(1a) is C₂₋₄ haloalkyl.
 8. A compound of claim 1, having the structure


9. A compound of claim 1, having the structure

or a pharmaceutically acceptable salt thereof.
 10. A compound of claim1, having the structure


11. A compound of claim 1, having the structure

or a pharmaceutically acceptable salt thereof.
 12. A compound of claim1, having the structure


13. A compound of claim 1, having the structure

or a pharmaceutically acceptable salt thereof.
 14. The compound of claim1, wherein said compound is selected from the group consisting of